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cDNA Isolation and Functional Characterization of UDP-D-glucuronic Acid 4-Epimerase Family from Ornithogalum caudatum Sen Yin † , Yu-Jia Sun † , Ming Liu, Li-Na Li and Jian-Qiang Kong * State Key Laboratory of Bioactive Substance and Function of Natural Medicines & Ministry of Health Key Laboratory of Biosynthesis of Natural Products, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China; [email protected] (S.Y.); [email protected] (Y.-J.S.); [email protected] (M.L.); [email protected] (L.-N.L.) * Correspondence: [email protected]; Tel.: +86-106-316-5169; Fax: +86-106-316-5197 † These authors contributed equally to this work. Academic Editor: Thomas J. Schmidt Received: 12 October 2016; Accepted: 6 November 2016; Published: 9 November 2016

Abstract: D-Galacturonic acid (GalA) is an important component of GalA-containing polysaccharides in Ornithogalum caudatum. The incorporation of GalA into these polysaccharides from UDP-D-galacturonic acid (UDP-GalA) was reasonably known. However, the cDNAs involved in the biosynthesis of UDP-GalA were still unknown. In the present investigation, one candidate UDP-D-glucuronic acid 4-epimerase (UGlcAE) family with three members was isolated from O. caudatum based on RNA-Seq data. Bioinformatics analyses indicated all of the three isoforms, designated as OcUGlcAE1~3, were members of short-chain dehydrogenases/reductases (SDRs) and shared two conserved motifs. The three full-length cDNAs were then transformed to Pichia pastoris GS115 for heterologous expression. Data revealed both the supernatant and microsomal fractions from the recombinant P. pastoris expressing OcUGlcAE3 can interconvert UDP-GalA and UDP-D-glucuronic acid (UDP-GlcA), while the other two OcUGlcAEs had no activity on UDP-GlcA and UDP-GalA. Furthermore, expression analyses of the three epimerases in varied tissues of O. caudatum were performed by real-time quantitative PCR (RT-qPCR). Results indicated OcUGlcAE3, together with the other two OcUGlcAE-like genes, was root-specific, displaying highest expression in roots. OcUGlcAE3 was UDP-D-glucuronic acid 4-epimerase and thus deemed to be involved in the biosynthesis of root polysaccharides. Moreover, OcUGlcAE3 was proposed to be environmentally induced. Keywords: UDP-D-glucuronic acid 4-epimerase; UDP-D-galacturonic acid; UDP-D-glucuronic acid; polysaccharides; Ornithogalum caudatum

1. Introduction UDP-D-glucuronic acid 4-epimerase, a very specific membrane-bound 4-epimerase, is able to catalyze the interconversion of UDP-GlcA and UDP-GalA (Scheme 1) [1–3]. The resultant UDP-GalA, the activated nucleotide sugar form of GalA, can serve as the precursor of various bacterial polysaccharides and plant pectins [3–5]. Also, UDP-GalA is the precursor of health-promoting galacturonides, providing GalA component for the synthesis of galacturonides [6–9]. By this token, UGlcAE played crucial role in the synthesis of GalA-containing molecules with potent activities. Therefore, many attentions had been directed to the study of UGlcAE. Thus far, UGlcAE activities had been detected in diverse prokaryotes including Bacillus cereus [4], Klebsiella pneumonia [10], Sinorhizobium meliloti [11], Escherichia coli [12] and Streptococcus pneumonia [13]. However, relatively less information was available on cDNA isolation and functional characterization of genes encoding

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UGlcAE fromMolecules plants, in monocots. Actually, the biochemical properties 2016, especially 21, x 2 of 16of UGlcAE from different organisms varied differentially. For example, some UGlcAE displayed substrate UGlcAE from plants, especially in monocots. Actually, the biochemical properties of UGlcAE from promiscuity [10], whileorganisms the others were substrate For specific [3–5,14,15]. Evendisplayed in plantsubstrate species, UGlcAEs different varied differentially. example, some UGlcAE [10],counterparts while the others were substratespecies specific [3–5,14,15]. Even in plantresults species, together UGlcAEs indicated in Arabidopsispromiscuity differ from in Poaceae [3,5,14,15]. These in Arabidopsis differ from counterparts in Poaceae species [3,5,14,15]. These results together indicated our understanding toward UGlcAE was far from enough. It is therefore necessary to strengthen the our understanding toward UGlcAE was far from enough. It is therefore necessary to strengthen the study of UGlcAE. characterization of of the encoding UGlcAE also a prerequisite to study Moreover, of UGlcAE. Moreover, characterization thegene gene encoding UGlcAE is also aisprerequisite to microbial production UDP-GlcA production by synthetic biology and and genetic engineering. Hence, an UGlcAEan UGlcAE microbial UDP-GlcA by synthetic biology genetic engineering. Hence, family containing three members was isolated from Ornithogalum caudatum in the present family containing three members was isolated from Ornithogalum caudatum in the present investigation. investigation.

O H

C

OH

O H

HO O

HO HO

H H

H

UGlcAE

C

UDP-GlcA

H

O H

HO

OH O-UDP

OH

H

H

H

OH O-UDP

UDP-GalA

Scheme 1. The interconversion of UDP-GlcA and UDP-GalA catalyzed by UglcAEs.

Scheme 1. The interconversion of UDP-GlcA and UDP-GalA catalyzed by UglcAEs.

Commented [J5]: We have change please confirm.

O. caudatum is a monocot containing four anti-cancer polysaccharides, in which D-GalA is one of the main monosaccharides [16]. It was well known that the incorporation of D-GalA into these O. caudatum is a monocot containing polysaccharides, in which D -GalA is one polysaccharides required the additionfour fromanti-cancer its activated form UDP-GalA, which was synthesized from UDP-GlcA by UGlcAE. Accordingly, UGlcAE was deemed to be highly expressed in O. into these of the main monosaccharides [16]. It was well known that the incorporation of D-GalA caudatum, which was in turn chosen as the start material for cDNA isolation of UGlcAE. polysaccharides required the addition from its activated form UDP-GalA, which was synthesized Here, three members of UGlcAE family, namely OcUGlcAE1, OcUGlcAE2 and OcUGlcAE3, from UDP-GlcA by UGlcAE. Accordingly, was deemed to be highlyreaction expressed O. caudatum, were isolated from O. caudatum basedUGlcAE on the RNA-Seq data. In vitro enzymatic verifiedin the interconversion function between UDP-GlcA and UDP-GalA of OcUGlcAE3. Importantly, their roles which was in turn chosen as the start material for cDNA isolation of UGlcAE. in GalA-containing polysaccharides biosynthesis were also discussed based on real-time quantitative Here, three members of UGlcAE family, namely OcUGlcAE1, OcUGlcAE2 and OcUGlcAE3, were PCR (RT-qPCR).

isolated from O. caudatum based on the RNA-Seq data. In vitro enzymatic reaction verified the 2. Results and between Discussion UDP-GlcA and UDP-GalA of OcUGlcAE3. Importantly, their roles interconversion function in GalA-containing polysaccharides were 2.1. Unigenes Retrieval from O. biosynthesis caudatum RNA-Seq Data also discussed based on real-time quantitative PCR (RT-qPCR). Transcriptome sequencing of O. caudatum was performed previously for the purpose of genes

isolation [17–19]. The resultant RNA-Seq data was thus aligned for functional annotation by Blast X database deposited in GenBank library in the present investigation. Six unigenes showing high similarity with UGlcAE, namely unigenes 26961, 32208, 57762, 34210, 61748 and 61778,from were retrieved (Table 1). Further ORF (open reading frame) Finder analyses toward these 2.1. Unigenes Retrieval O. caudatum RNA-Seq Data unigenes showed unigene 57762 contained a complete ORF encoding UGlcAE, while the other five unigenessequencing harbored partial sequence. Specifically, unigene57762for wasthe 2247purpose bp long Transcriptome ofUGlcAE–encoding O. caudatum was performed previously of genes with an ORF of 1398 bp encoding 465 aa and consisted of a partial 5’-untranslated region (UTR) of isolation [17–19]. The resultant RNA-Seq data was thus aligned for functional annotation by Blast X 270 bp and 3’-UTR of 579 bp. Unigenes 26961 and 61778 were 1011 and 478 bp long, respectively. against public Both protein database GenBank library inUnigenes the present Six unigenes of them displayeddeposited similarity toin 5’-end of an UGlcAE gene. 32208 investigation. and 61748 were 634 Commented [J6]: Italic or not? Uni 664 bp inwith length,UGlcAE, both exhibiting sequence identity to 3’-end of an UglcAE gene.34210, Unigene61748 34210 and 61778, showing high and similarity namely unigenes 26961, 32208, 57762, the paper. was 383 bp long and showed sequence similarity to a middle region of an UglcAE gene. These data were retrievedtogether (Tablerevealed 1). Further ORF (open reading frame) Finder analyses toward these unigenes that there were at least three members in O. caudatum UGlcAE family. The detailed showed unigene 57762 contained complete ORF encoding UGlcAE, while the other five unigenes sequence information of theasix unigenes was summarized in Table 1. public protein 2. Results andagainst Discussion

harbored partial UGlcAE–encoding sequence. Specifically, unigene 57762 was 2247 bp long with an ORF of 1398 bp encoding 465 aa and consisted of a partial 5’-untranslated region (UTR) of 270 bp and 3’-UTR of 579 bp. Unigenes 26961 and 61778 were 1011 and 478 bp long, respectively. Both of them displayed similarity to 5’-end of an UGlcAE gene. Unigenes 32208 and 61748 were 634 and 664 bp in length, both exhibiting sequence identity to 3’-end of an UGlcAE gene. Unigene 34210 was 383 bp long and showed sequence similarity to a middle region of an UGlcAE gene. These data together revealed that there were at least three members in O. caudatum UGlcAE family. The detailed sequence information of the six unigenes was summarized in Table 1. 2.2. cDNA Isolation of OcUGlcAE Family To verify the authenticity of these unigenes, PCR amplification of these sequences from O. caudatum by a nested PCR assay using gene-specific primers was performed. Unigenes 26961 and 32208 displayed high similarity with 5’-end and 3’-end of an UGlcAE gene respectively, suggesting

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the two unigenes were likely to locate on the same UglcAE gene. To test this notion, the 5’-end primers of unigene 26961 were paired with the 3’-end primers of unigene 32208 to amplify an UglcAE gene Molecules 2016, 21, 1505 3 of 15 using O. caudatum cDNA as the template. After two rounds PCR, a specific PCR fragment, designated TM as OcUGlcAE1, was thus generated and ligated to*yield the 61748 664 into pEASY N -Blunt 354 310recombinant plasmid pEASY-OcUGlcAE1 for61778 sequencing. Results showed that the amplified 1320 bp fragment showed 100% 478 148 330 * N sequence identity with unigenes 26961 and 32208, suggesting the cDNA was a bona fide UGlcAE gene N, no sequences; *, partial ORF sequence in O. caudatum genome (Figure 1). 2.2. cDNA Isolation of OcUGlcAE Family Table 1. Unigenes sharing high sequence similarity with UDP-D-glucuronic acid 4-epimerase. To verify the authenticity of these unigenes, PCR amplification of these sequences from O. caudatum by a nested PCR assay using gene-specific primers was performed. Unigenes 26961 and Sequence Structure (bp) ID with Length (bp) 32208 displayed highUnigene similarity 5’-end and 3’-end of an UGlcAE gene respectively, suggesting 5’-UTR ORF 3’-UTR the two unigenes were likely to locate on the same UglcAE gene. To test this notion, the 5’-end 1011 the 3’-end206 * N primers of unigene 2696126961 were paired with primers 805 of unigene 32208 to amplify an UglcAE 32208 634 N 459 * 175 gene using O. caudatum57762 cDNA as the 2247 template. After PCR,579 a specific PCR fragment, 270 two rounds 1398 designated as OcUGlcAE1, was thus generated and ligated 34210 383 N into pEASY 383 * TM-Blunt N to yield the recombinant 61748 for sequencing. 664 * the amplified 310 plasmid pEASY-OcUGlcAE1 ResultsNshowed354 that 1320 bp fragment 61778 478 148 330 * showed 100% sequence identity with unigenes 26961 and 32208, suggestingNthe cDNA was a bona fide N, no sequences; UGlcAE gene in O. caudatum genome (Figure 1).*, partial ORF sequence.

OcUGlcAE1

ORF

ATG 5'-UTR Unigene 26961

OcUGlcAE2

ATG 5'-UTR

TGA 3'-UTR Unigene 32208

ORF

TAA 3'-UTR

Unigene 57762

OcUGlcAE3

ATG 5'-UTR Unigene 61778

ORF

TGA 3'-UTR

Unigene 34210

Unigene 61748

Figure 1. Schematic diagram of OcUGlcAE family in O. caudatum. Figure 1. Schematic diagram of OcUGlcAE family in O. caudatum.

The full-length ORF corresponding to unigene 57762 was directly isolated from O. caudatum. Theresultant full-length ORF corresponding to unigene was directly isolatedwhich from were O. caudatum. The cDNA was 1398 bp in length and was57762 designated to OcUGlcAE2, then TM-Blunt Theinserted resultant 1398tobp in length and was designated to OcUGlcAE2, for which were then intocDNA pEASYwas generate a recombinant plasmid pEASY-OcUGlcAE2 sequencing. inserted into pEASY -Blunt tothat generate a recombinant plasmid pEASY-OcUGlcAE2 Sequencing resultsTM indicated the cDNA was identical to the unigene 577629 (Figure for 1). sequencing. Unigenes 61778 and 61748 exhibited high similarity to the 5’and 3’-end of an UGlcAE Sequencing results indicated that the cDNA was identical the unigene 577629 (Figure 1). gene, which provided clue 61748 that the two unigenes might be located in and the same gene. the pair-wise Unigenes 61778a and exhibited high similarity to the 5’3’-end of anThus, UGlcAE gene, which primers corresponding to unigenes were used to amplify the third provided a clue that the two unigenes61778 mightand be 61748 located in the same gene. Thus, theOcUGlcAE pair-wise gene primers from O. caudatum. The nested PCR led to the occurrence of a specific fragment of 1389 bp encoding corresponding to unigenes 61778 and 61748 were used to amplify the third OcUGlcAE gene from 462 aa. This was sequenced with the form ofofpEASY-OcUGlcAE3, a aa. O. caudatum. The full-length nested PCRORF led to the then occurrence of a specific fragment 1389 bp encoding 462 pEASYTM-Blunt derived plasmid. Result verified the 100% identity of OcUGlcAE3 to unigenes TM 61778 This full-length ORF was then sequenced with the form of pEASY-OcUGlcAE3, a pEASY -Blunt and 61748. Interestingly, OcUGlcAE3 was also 100% identical to unigene 34210. These data together derived plasmid. Result verified the 100% identity of OcUGlcAE3 to unigenes 61778 and 61748. revealed that unigenes 61778, 61748 and 34210 located on the same OcUGlcAE3 gene. Therefore, Interestingly, OcUGlcAE3 was also 100% identical to unigene 34210. These data together revealed that there were at least three members in O. caudatum UGlcAE family (Figure 1). The three full-length unigenes 61778, 61748 andhence 34210deposited located on same OcUGlcAE3 Therefore, there were at least OcUGlcAE genes were in the GenBank library with gene. accession numbers of KX429689, three members in O. caudatum UGlcAE family (Figure 1). The three full-length OcUGlcAE genes were KX429690 and KX429691 (Table 2).

hence deposited in GenBank library with accession numbers of KX429689, KX429690 and KX429691 (Table 2).

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Table 2. Comparison of the predicted OcUGlcAE proteins as deduced from their cDNA sequences.

Table 2. Comparison of the predicted OcUGlcAE proteins as deduced from their cDNA sequences. Gene Gene OcUGlcAE1 OcUGlcAE1 OcUGlcAE2 OcUGlcAE2 OcUGlcAE3 OcUGlcAE3

Nucleotide Nucleotide Corresponding Accession Accession Corresponding Unigene (s) Number Number Unigene (s) Unigenes 26961 Unigenes 26961 KX429689 KX429689 and 32208 and 32208 KX429690 57762 KX429690 Unigene Unigene 57762 Unigenes 61778, Unigenes 61778, KX429691 KX429691 61748 and 34210 61748 and 34210

Length ORF Length (bp) ORF a.a. (bp) a.a. (bp) (bp)

kDa kDa

Protein Protein Identity (%) pI Identity (%) pI OcUGlcAE2 OcUGlcAE3 OcUGlcAE2 OcUGlcAE3

1701 1701 1320 1320 439 439 48.462 48.462

9.66 9.66

74 74

76 76

2247 2247 1398 1398 465 465 50.057 50.057 1847 1389 463 50.579 1847 1389 463 50.579

9.76 9.76 9.77 9.77

100 100 77 77

77 77 100 100

2.3. Bioinformatics Bioinformatics Characterization Characterization of of OcUGlcAE OcUGlcAE Family 2.3. Family UGlcAE belongs the amino acid sequences of UGlcAE belongs to to one onemember memberofofthe theSDR SDRenzyme enzymefamilies. families.Hence, Hence, the amino acid sequences OcUGlcAE proteins contained two conserved motifs widely existed in SDR protein families. The first of OcUGlcAE proteins contained two conserved motifs widely existed in SDR protein families. The + (Figure 2). one was N-terminal GxxGxxG motif (xmotif = any(xamino for acid) the binding NAD (P) first one an was an N-terminal GxxGxxG = anyacid) amino for thetobinding to NAD (P)+ The second conserved motif referred to the catalytic YxxxK motif (where x represents any amino acid) (Figure 2). The second conserved motif referred to the catalytic YxxxK motif (where x represents (Figure 2). acid) (Figure 2). any amino

Figure 2. Sequence alignments of OcUGlcAE family. The red pentagram indicated the conserved Figure 2. Sequence alignments of OcUGlcAE family. The red pentagram indicated the conserved motifs in OcUGlcAE family. motifs in OcUGlcAE family.

Next, the most probable locations of trans-membrane helices in the three sequences were Next, by theTMHMM most probable locations of trans-membrane helices in the three sequences predicted predicted program (http://www.cbs.dtu.dk/services/TMHMM/) [20]. Aswere shown in the by TMHMM program (http://www.cbs.dtu.dk/services/TMHMM/) [20]. As shown in the supporting supporting material Figure S1, all the three proteins were trans-membrane proteins, which was material Figure the threepublications proteins were trans-membrane proteins, wasOcUGlcAE1 consistent with consistent with S1, theallprevious [3,14,15]. Among the three which proteins, and the previous publications [3,14,15]. Among the three proteins, OcUGlcAE1 and OcUGlcAE3 both had OcUGlcAE3 both had two trans-membrane helices with more than 80% probability, while OcUGlcAE2 two trans-membrane helices with more thanwith 80% more probability, whileprobability. OcUGlcAE2 containedthe only one contained only one trans-membrane helice than 80% Moreover, region trans-membrane with more than 80%probability probability.toMoreover, region between 110 and 150 aa between 110 andhelice 150 aa had about 35% form the the second trans-membrane helice in had about 35% probability to form the second trans-membrane helice in OcUGlcAE2 (Figure S1). OcUGlcAE2 (Figure S1). Phylogenetic relationships relationships of of OcUGlcAE OcUGlcAE with with other -glucose 4-epimerase) Phylogenetic other UGlcAE, UGlcAE, UGE UGE (UDP(UDP-D D-glucose 4-epimerase) and UXE -xylose 4-epimerase) enzymes were analyzed using the neighbor-joining method and UXE (UDP(UDP-D D-xylose 4-epimerase) enzymes were analyzed using the neighbor-joining method (Figure 3). As indicated in Figure 4, UGlcAE, UGE and UXE belonged to three distinct clades, suggesting the divergent functions among the tree enzymes were present through evolution. The

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30 11 88 33 11 CD WW GG 11 11 00 22 WW GG mm uu a ii t rr a ee 55 r tt 55 y cc 77 l p a a p u 00 5 4 bb u m 0 . o 07 5 4 m o uu p aa 7 M rp r 7 t s G i i 0 u s A G C C b m at r r 0 o a i C e C s C u t u m t a T ee r c e sAu aG Ti7n s aa a tt ac ct A u A p m l i c c qCest a e ac a u sri uaaa rl a a a r t b t iena n ssrCnie c tnnt au o bb T r c a s t a o i o c a a t us a sf ee i p iu ip bm s wnc ii tAd ee r oa a g os nl s e i cll an a uq sn u yshc ar ey e na r Jo J q rrs b nge eo c l naalacn r c e i o a u o u o i aii uhh r boeoonreoW nt p a W u a o g w r r a r o chb rttsu ic aaJ v ttxq deagallb a v ass e c n c g mt nts icttn i snn gu mmr i udi ao sslru s au au a o t u o a ooc o is oj u f r gxtv ux sg lcl min ipau gs a x t ualta otxgl op soh aa l uad aa siai s e pp erse ry cc ls amaaamvdi o dplcm inst s g l h m l u u i u m bs o m e id a i a e ia a usd as pd boo os z ffcufuuoloogimdd oz id ru r lh eoo iar tPe eb y ooyxoxlrarhopunn y e E hG bra nM r rrsereabgbthyeaa l t a h l M M r d i h P t o 1 s A o o T1 a g J 1P 1a C 1O is z O hc 1s rr 1a. rC1 nn 1fcfo eh .. 11 er 1. .1 y 1 c AA .j8 hth yy.oyoh 7 o3.7 oo1. .. .6 1251ZE3 634a C 1 .r .2ss5rsrtTiTOrGH.8 3 0 8 h O 1 5 O P h 3 1 2 1 81 11 9 1E7EPP0c1 ci1n1111103 .179001A557 9. 04 8 52.9502.c940.A0A111y.yn.r.....89 7 0 . 4 c l 3 6 . 2 4 O 2 3 21 3 6 9 950027 95 21 346G0999c9l582s3sr51 87 4 53 2 O 8 9 4 5 1 47 .53 0 09 867980244U0756l5G700P6P10 3 C QQ 1 59 5 5 U 8 803 7106 .41 01122038c18C0GB KK .217 0 9 U 7 . 1 6K B 8 1 c 8 2C 4C3 0017 07 K 5cAO 3G 1901005O00A 608371 Z 050 6 0 000P 1 0 C O 73 UU 4405 P M 7NCHB P B P 8 FFP531 PPPYPPPPPXPK X 3030ANKA 7 A X X A W A XNXKXNNXX 30C0 A 608 C N U0Y NPAP FPM AW EAX X

6 of 15 (Figure 3). As indicated in Figure 4, UGlcAE, UGE and UXE belonged to three distinct clades, suggesting UGlcAE family could beamong divided groups, which included diverse UGlcAEs from eudicots, the divergent functions theinto treethree enzymes were present through evolution. The UGlcAE family Next, these E. coli crude extract expressing trun-OcUGlcAE proteins were applied to examine their monocots and bacteria, respectively. Although OcUGlcAE1~3 werefrom most similar monocots to monocots could be divided into three groups, which included diverse UGlcAEs eudicots, and activities in a total reaction volume of 100 μL. After incubation at 30 °C for 30 min, twenty microlitres UGlcAE, the three UGlcAE enzymes belonged towere different (Figure 3).UGlcAE, The evolutionary bacteria, respectively. Although OcUGlcAE1~3 most subgroups similar to monocots the three supernatant was directly analyzed by HPLC. As illustrated in Figure S3, there are no specific peaks differences amongbelonged the three OcUGlcAE enzymes may 3). provide a clue thatdifferences the threeamong enzymes UGlcAE enzymes to different subgroups (Figure The evolutionary the corresponding reaction products in the three reaction mixtures, suggesting no activities toward functioned differentially. three OcUGlcAE enzymes may provide a clue that the three enzymes functioned differentially. UDP-GlcA occurred in these OcUGlcAE proteins (Figure S3). The unavailability of active recombinant enzymes was present when trun-OcUGlcAEs were expressed in E. coli, presumably due to the N-terminal truncation that resulted in improper folding and decreased stability of these recombinant proteins. Three full-length OcUGlcAE proteins were therefore heterologously expressed in P. pastoris Eudicots for active recombinant enzymes.

2.5. Heterologous Expression of OcUGlcAEs in P. pastoris

For analysis of enzymatic activity of OcUGlcAEs, the supernatant of three recombinant P. pastoris strains containing pPIC3.5kOcUGlcAEs was used directly as the biocatalyst for the interconversion of Monocots UGlcAE UDP-GalA and UDP-GlcA, respectively. The newly formed products were monitored by a HPLC-based assay. When UDP-GlcA and NAD+ were incubated with the supernatant harboring a recombinant OcUGlcAE3 protein, a new peak, having the same retention time and UV spectra as the authentic UDP-GalA, was detected in the HPLC profile (FigureBacteria 4). The supernatant of P. pastoris expressing the empty control vector did not yield a new peak, indicating the observed new peak was exclusively produced by the recombinant OcUGlcAE3. To further verify that the newly formed reaction product representing exactly UDP-GalA, the UDP-GalA standard wasUGE co-injected with the reaction mixture under the HPLC-based assay. Only a UDP-GalA peak with no shouldering was UXE of UDP-GlcA found by HPLC analysis, suggesting UDP-GalA was produced from 4-epimerization (Figure S4). Finally, to determine the identity of the newly formed product, the enzymatic product eluting from the HPLC column was collected and confirmed by mass spectrometry. The mass 0.2 spectrometer gave the most abundant ion at m/z 603.01874, corresponding to an adduct of the detected molecule with a tree sodium [M +OcUGlcAE Na]+. Thefamily molecular massUGlcAEs, of the epimerized Figure 3. Phylogenetic of O.atom caudatum OcUGlcAE with UGlcAEs, UGEs UXEs Figure 3. Phylogenetic tree of O. caudatum family with other other UGEs and and derivative UXEs from varied organisms. The tree was constructed by MEGA 7.0 using Neighbor-joining method. of UDP-GalA catalyzed by OcUGlcA3 was thus inferred to be 580, which was identical toThe that of from varied organisms. The tree was constructed by MEGA 7.0 using Neighbor-joining method. reliability of the tree was measured by bootstrap analysis with 1000 replicates. Red circles represent UDP-GalA standard (Figure 5A). Taken together, these data unambiguously identified that The reliability of the tree was measured by bootstrap analysis with 1000 replicates. Red circles represent the the member of family. Branch lengths are are shown to toproteins scale. newly catalyzed byBranch recombinant OcUGlcA3 was UDP-GalA. theformed memberproduct of OcUGlcAE OcUGlcAE family. lengths shown scale. 2.4. Bacterial Expression of OcUGlcAEs The three OcUGlcAE enzymes were predicted to be membrane-bound proteins by TMHMM algorithm (Figure S1). Recombinant membrane-associated proteins were shown to be difficult to express UDP-GlcA + and purify in E. coli, presumably due to membrane-spanning domains [17,21]. The truncated NAD OcUGlcAEs, lacking the putative trans-membrane motifs, were thus generated for soluble expressions NADH in E. coli. Specifically, the trans-membrane domains of the three proteins were deleted from the N-terminus to form three truncated versions, namely OcUGlcAE1 (Δ1–116), OcUGlcAE2 (Δ1–136) and OcUGlcAE3 (Δ1–128). Then, these N-terminal truncated OcUGlcAE genes were inserted into EcoRI/Hind III linearized pET-28a (+) to yield three recombinant plasmids, pET28aOcUGlcAE1 (Δ1–116), pET28aOcUGlcAE2 (Δ1–136) and pET28aOcUGlcAE3 (Δ1–128) (Table S1). Truncated OcUGlcAE proteins (trun-OcUGlcAEs) were then induced to be heterologously expressed in Transetta (DE3) with the form of pET28aOcUGlcAE1 (Δ1–116), pET28aOcUGlcAE2 (Δ1–136) and pET28aOcUGlcAE3 (Δ1–128), respectively. SDS-PAGE analyses showed these truncated proteins were mainly appeared as insoluble inclusion bodies, suggesting the improper folding of these trun-OcUGlAE proteins occurred. It was well known that molecular chaperones were able to assist the refolding of inclusion body [22,23]. a chaperone plasmid pGro7 was co-expressed with P. each of the three plasmids 4. Hence, pastoris expressing the Figure 4. HPLC analyses of the products formed by the supernatant from P. pastoris expressing the containing truncated OcUGlcAE genes in E. coli. The chaperone plasmid pGro7 contained +. The two + empty vector alone (1) or pPIC3.5kOcUGlcAE3 (2) in the presence of UDP-GlcA and NAD empty vector alone (1) or pPIC3.5kOcUGlcAE3 (2) in the presence of UDP-GlcA and NAD . The newly genes, groES and groEL, encoding for chaperone proteins. GroES and GroEL were well known to newly formed UDP-GalA was marked a red arrow. formed UDP-GalA was marked by a redbyarrow. work in cooperation in the folding process [22,23]. With the help the chaperone proteins GroES and GroEL, the recovery of the soluble trun-OcUGlcAE proteins from inclusion bodies increased, as shown in the supporting material Figure S2.

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2.4. Bacterial Expression of OcUGlcAEs The three OcUGlcAE enzymes were predicted to be membrane-bound proteins by TMHMM algorithm (Figure S1). Recombinant membrane-associated proteins were shown to be difficult to express and purify in E. coli, presumably due to membrane-spanning domains [17,21]. The truncated OcUGlcAEs, lacking the putative trans-membrane motifs, were thus generated for soluble expressions in E. coli. Specifically, the trans-membrane domains of the three proteins were deleted from the N-terminus to form three truncated versions, namely OcUGlcAE1 (∆1–116), OcUGlcAE2 (∆1–136) and OcUGlcAE3 (∆1–128). Then, these N-terminal truncated OcUGlcAE genes were inserted into EcoRI/Hind III linearized pET-28a (+) to yield three recombinant plasmids, pET28aOcUGlcAE1 (∆1–116), pET28aOcUGlcAE2 (∆1–136) and pET28aOcUGlcAE3 (∆1–128) (Table S1). Truncated OcUGlcAE proteins (trun-OcUGlcAEs) were then induced to be heterologously expressed in Transetta (DE3) with the form of pET28aOcUGlcAE1 (∆1–116), pET28aOcUGlcAE2 (∆1–136) and pET28aOcUGlcAE3 (∆1–128), respectively. SDS-PAGE analyses showed these truncated proteins were mainly appeared as insoluble inclusion bodies, suggesting the improper folding of these trun-OcUGlAE proteins occurred. It was well known that molecular chaperones were able to assist the refolding of inclusion body [22,23]. Hence, a chaperone plasmid pGro7 was co-expressed with each of the three plasmids containing truncated OcUGlcAE genes in E. coli. The chaperone plasmid pGro7 contained two genes, groES and groEL, encoding for chaperone proteins. GroES and GroEL were well known to work in cooperation in the folding process [22,23]. With the help the chaperone proteins GroES and GroEL, the recovery of the soluble trun-OcUGlcAE proteins from inclusion bodies increased, as shown in the supporting material Figure S2. Next, these E. coli crude extract expressing trun-OcUGlcAE proteins were applied to examine their activities in a total reaction volume of 100 µL. After incubation at 30 ◦ C for 30 min, twenty microlitres supernatant was directly analyzed by HPLC. As illustrated in Figure S3, there are no specific peaks corresponding reaction products in the three reaction mixtures, suggesting no activities toward UDP-GlcA occurred in these OcUGlcAE proteins (Figure S3). The unavailability of active recombinant enzymes was present when trun-OcUGlcAEs were expressed in E. coli, presumably due to the N-terminal truncation that resulted in improper folding and decreased stability of these recombinant proteins. Three full-length OcUGlcAE proteins were therefore heterologously expressed in P. pastoris for active recombinant enzymes. 2.5. Heterologous Expression of OcUGlcAEs in P. pastoris For analysis of enzymatic activity of OcUGlcAEs, the supernatant of three recombinant P. pastoris strains containing pPIC3.5kOcUGlcAEs was used directly as the biocatalyst for the interconversion of UDP-GalA and UDP-GlcA, respectively. The newly formed products were monitored by a HPLC-based assay. When UDP-GlcA and NAD+ were incubated with the supernatant harboring a recombinant OcUGlcAE3 protein, a new peak, having the same retention time and UV spectra as the authentic UDP-GalA, was detected in the HPLC profile (Figure 4). The supernatant of P. pastoris expressing the empty control vector did not yield a new peak, indicating the observed new peak was exclusively produced by the recombinant OcUGlcAE3. To further verify that the newly formed reaction product representing exactly UDP-GalA, the UDP-GalA standard was co-injected with the reaction mixture under the HPLC-based assay. Only a UDP-GalA peak with no shouldering was found by HPLC analysis, suggesting UDP-GalA was produced from 4-epimerization of UDP-GlcA (Figure S4). Finally, to determine the identity of the newly formed product, the enzymatic product eluting from the HPLC column was collected and confirmed by mass spectrometry. The mass spectrometer gave the most abundant ion at m/z 603.01874, corresponding to an adduct of the detected molecule with a sodium atom [M + Na]+ . The molecular mass of the epimerized derivative of UDP-GalA catalyzed by OcUGlcA3 was thus inferred to be 580, which was identical to that of UDP-GalA standard (Figure 5A). Taken together, these data unambiguously identified that the newly formed product catalyzed by recombinant OcUGlcA3 proteins was UDP-GalA.

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A A

B B

[M+Na]+ [M+Na]+

[M+Na]+ [M+Na]+

[M+K]+ [M+K]+

Figure 5. Electrospray positive ion mass spectra for UDP-GalA (A) and UDP-GlcA (B). Figure 5. Electrospray positive ion mass spectra for UDP-GalA (A) and UDP-GlcA (B). Figure 5. Electrospray positive ion mass spectra for UDP-GalA (A) and UDP-GlcA (B).

Next, the ability of the recombinant OcUGlcAE3 to convert UDP-GalA to UDP-GlcA was also Next, the the ability in the recombinant OcUGlcAE3 to convert UDP-GalA to UDP-GlcA was also Next, ability of Figure recombinant OcUGlcAE3 to convert UDP-GalA UDP-GlcA was confirmed. As shown 6, incubation of the supernatant from P. pastoris to expressing OcUGlcAE3 confirmed. As shown in Figure 6, incubation of the supernatant from P. pastoris expressing OcUGlcAE3 confirmed. As shown in Figure 6, incubation supernatantwhich from P. with UDP-GalA resulted in the generationofoftheUDP-GlcA, was determined by co-injection UDP-GalA resulted UDP-GlcA, co-injection with UDP-GalA resulted in the generation of which was determined by co-injection analysis (Figure S5) and mass spectrometry (Figure 5B) as mentioned above. analysis (Figure S5) and mass spectrometry (Figure 5B) as mentioned above. analysis 500 500

Absorbance [mAU] Absorbance [mAU]

400 400 300 300

NAD+ NAD+

200 200

NADH NADH NADH NADH

UDP-GalA UDP-GalA UDP-GlcA UDP-GlcA UDP-GalA UDP-GalA

100 100 -10 -10 0.0 0.0

5.0 5.0

10.0 15.0 20.0 10.0Retention 15.0Time [min] 20.0 Retention Time [min]

25.0 25.0

2 2 1 1

30.0 30.0

Figure 6. HPLC analyses of reaction mixture containing the supernatant from GS115 (1) or P. pastoris Figure analyses of reaction mixture the Figure 6. HPLC HPLC analyses(2) ofusing reaction mixture containing containing the supernatant supernatantfrom from GS115 GS115(1) (1)or orP. P. pastoris pastoris expressing OcUGlcAE3 UDP-GalA as the substrate. expressing expressing OcUGlcAE3 (2) using UDP-GalA as the substrate.

Besides the supernatant, the microsomal fraction from P. pastoris expressing OcUGlcAE3 was Besides the supernatant, microsomal from P. pastoris expressing OcUGlcAE3 was observed to have 4-epimerase the activity capable fraction of interconverting UDP-GalA and UDP-GlcA (Figure observed to have 4-epimerase activity capable of interconverting UDP-GalA and UDP-GlcA (Figure

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Besides the supernatant, the microsomal fraction from P. pastoris expressing OcUGlcAE3 was 7). Thesetodata thatactivity the recombinant OcUGlcAE3 can form microsomal protein by the observed haveindicated 4-epimerase capable of interconverting UDP-GalA and UDP-GlcA (Figure 7). N-terminal membrane anchor sequence, or can be secrete into the extracellular space through the These data indicated that the recombinant OcUGlcAE3 can form microsomal protein by the N-terminal guidance ofanchor the signal peptide.orMoreover, OcUGlcAE3 not require exogenous NAD , suggesting the membrane sequence, can be secrete into the did extracellular space through the +guidance of the + + , suggesting recombinant contained tightly NAD , which was consistentthe with previous signal peptide.OcUGlcAE3 Moreover, OcUGlcAE3 did not bound require exogenous NAD recombinant reports [3,5,15]. OcUGlcAE3 contained tightly bound NAD+ , which was consistent with previous reports [3,5,15].

UDP-GalA NADH

UDP-GlcA

NAD+

P. pastoris. pastoris. 1, HPLC Figure 7. HPLC profile of reaction mixture catalyzed by microsome fractions from P. profile of microsomes microsomes from ofof microsomes from P. profile from P. P. pastoris pastoris expressing expressingOcUGlcAE3. OcUGlcAE3.2,2,HPLC HPLCanalysis analysis microsomes from +. 3, HPLC + . 3, chromatograph of reaction mixture pastoris expressing OcUGlcAE3 in the of NAD P. pastoris expressing OcUGlcAE3 in presence the presence of NAD HPLC chromatograph of reaction containing microsomes from P.from pastoris expressing OcUGlcAE3 in theinpresence of UDP-GlcA and mixture containing microsomes P. pastoris expressing OcUGlcAE3 the presence of UDP-GlcA + + NADNAD . 4, HPLC profileprofile of reaction mixture containing microsomes from P.from pastoris expressing empty and . 4, HPLC of reaction mixture containing microsomes P. pastoris expressing + . 5, HPLC 5, HPLC analysis of microsomes from GS115 vector vector alone in the presence of UDP-GlcA and NAD empty alone in the presence of UDP-GlcA and+.NAD analysis of microsomes from +. 6 and +7.refer to7HPLC separation of the standard (6) and UDP-GalA in the presence of NADof GS115 in the presence NAD 6 and refer to HPLC separation of theUDP-GlcA standard UDP-GlcA (6) and UDP-GalA (7), respectively. (7), respectively.

On the the contrary, contrary, the the 4-epimerase 4-epimerase activity activity was was not not detected detected either either in in the the supernatant supernatant or or microsomal microsomal On fraction from P. pastoris expressing OcUGlcAE1 or OcUGlcAE2. fraction from P. pastoris expressing OcUGlcAE1 or OcUGlcAE2. 2.6. Biochemical Properties of OcUGlcAE3 OcUGlcAE3 OcUGlcAE3 was a relatively broad pH range between 4 and 411and (Figure 8A). The8A). pH wasactive activeover over a relatively broad pH range between 11 (Figure value OcUGlcAE3 was shown as an “M” 8A). OcUGlcAE3 retained no more The pHcurve valueofcurve of OcUGlcAE3 was shown as antype “M”(Figure type (Figure 8A). OcUGlcAE3 retained no than 10% whenwhen the pH was was 4.0. The enzymatic activity increased gradually withwith the more thanactivity 10% activity thevalue pH value 4.0. The enzymatic activity increased gradually increase of pH. When pHpH reached thethe range the increase of pH. When reached rangebetween between8 8and and8.5, 8.5,the theenzyme enzyme displayed displayed the highest activity. With the further increase of pH to about 9, the activity activity. activity of of the the enzyme enzyme decreased decreased slightly. slightly. And then, as the pH increased from 9 to 10, the activity of OcUGlcAE3 increased gradually, and And then, as the pH increased from 9 to 10, the activity of OcUGlcAE3 increased gradually, and reached reached the highest activity again at pH value of 10. Next, with the further increase of pH, the highest activity again at pH value of 10. Next, with the further increase of pH, the activitythe of activity of OcUGlcAE3 began When to decrease. When12, pH reached 12, OcUGlcAE3 kept only 80% ofpH its OcUGlcAE3 began to decrease. pH reached OcUGlcAE3 kept only 80% of its activity. The activity. pH profile of OcUGlcAE3 showed type, which with [14,15]. that of profile of The OcUGlcAE3 showed an “M” type, which an was“M” inconsistent withwas that inconsistent of other UGlcAEs other UGlcAEs [14,15]. the exact was reason thisthe discrepancy was notofclear, the alkaline Although the exact reasonAlthough of this discrepancy not of clear, alkaline preference OcUGlcAE3 was preference of OcUGlcAE3 was certain, which was also in other UGlcAEs, like and Arabidopsis certain, which was also observed in other UGlcAEs, likeobserved Arabidopsis UGlcAEs [3,14,15] Poaceae UGlcAEs [5]. [3,14,15] and Poaceae UGlcAEs [5]. UGlcAEs OcUGlcAE3 displayed displayed activity activity over over aa broad broad temperature temperature range range between between 00 ◦°C and 60 60 ◦°C and OcUGlcAE3 C and C and ◦ had maximal activity at 42 °C (Figure 9B). The temperature profile of OcUGlcAE also showed “M” had maximal activity at 42 C (Figure 9B). The temperature profile of OcUGlcAE also showed “M” type (Figure (Figure 8B). type 8B). The The activity activity of of OcUGlcAE3 OcUGlcAE3gradually graduallyrose rosewith withthe theincrease increaseofoftemperature temperaturefrom from0 ◦ ◦ C. 3030 °C. C. OcUGlcAE3 activity reached the the second highhigh levellevel whenwhen the temperature was 30was °C. 30 Then, 0toto OcUGlcAE3 activity reached second the temperature with the further increase of temperature, the activity of OcUGlcAE3 began to decrease. When the Then, with the further increase of temperature, the activity of OcUGlcAE3 began to decrease. When ◦ temperature reached 37 °C,37 OcUGlcAE3 was only 70%only activity. the further increase of temperature, the temperature reached C, OcUGlcAE3 was 70%With activity. With the further increase of ◦ C. temperature OcUGlcAE3 activity beganactivity to increase and reached the at 42 °C.level Next, temperature, OcUGlcAE3 began to increase andhighest reachedlevel the highest at as 42 the Next, as the ◦ C to ◦ C, the activity increased from 42 °Cfrom to 60 the of OcUGlcAE3 decreased significantly. When the temperature increased 42 °C, 60 activity of OcUGlcAE3 decreased significantly. When ◦ temperature reached 65 °C, only kept (Figure(Figure 8B). Further kinetic the temperature reached 65OcUGlcAE2 C, OcUGlcAE2 only less keptthan less10% thanactivity 10% activity 8B). Further analyses indicated that OcUGlcAE3 had an apparent Km of 0.17 ± 0.07 mM for UDP-D-Glc, which was comparable with that of Arabidopsis UGlcAEs [3].

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kinetic analyses indicated that OcUGlcAE3 had an apparent Km of 0.17 ± 0.07 mM for UDP-D-Glc, which was comparable with that of Arabidopsis UGlcAEs [3]. Molecules 2016, 21, 1505 9 of 15

A

B

Figure 8. The effect of pH (A) or temperature (B) on the OcUGlcAE3 activity. Figure 8. The effect of pH (A) or temperature (B) on the OcUGlcAE3 activity.

2.7. Expression Analyses of OcUGlcAE in O. caudatum 2.7. Expression Analyses of OcUGlcAE in O. caudatum OcUGlcAE regulated the biosynthesis of UDP-GalA, a monosaccharide donor for the synthesis OcUGlcAE regulated the biosynthesis of UDP-GalA, a monosaccharide donor for the synthesis of GalA-containing polysaccharides in O. caudatum. OcUGlcAE was thus proposed to be responsible for of GalA-containing polysaccharides in O. caudatum. OcUGlcAE was thus proposed to be responsible the biosynthesis these GalA-containing polysaccharides. To probe the relevance of OcUGlcAE to for the biosynthesis these GalA-containing polysaccharides. To probe the relevance of OcUGlcAE to polysaccharides biosynthesis, expression profiles of the three genes in varied tissues of O. caudatum polysaccharides biosynthesis, expression profiles of the three genes in varied tissues of O. caudatum were hence performed based on RT-qPCR results (Figure 9). The transcript level of OcUGlcAE3 was were hence performed based on RT-qPCR results (Figure 9). The transcript level of OcUGlcAE3 was most abundant in roots, where OcUGlcAE3 mRNA levels were almost 2-fold higher than that in most abundant in roots, where OcUGlcAE3 mRNA levels were almost 2-fold higher than that in sterile bulbs of O. caudatum (Figure 9). This root-specific expression pattern of OcUGlcAE3 revealed sterile bulbs of O. caudatum (Figure 9). This root-specific expression pattern of OcUGlcAE3 revealed its involvement in the biosynthesis of root polysaccharides containing uronic acids. The sterile its involvement in the biosynthesis of root polysaccharides containing uronic acids. The sterile bulbs, cultivated in sterile 6,7-V medium under a controlled condition, were the same organ as O. bulbs, cultivated in sterile 6,7-V medium under a controlled condition, were the same organ as caudatum bulbs, which were grown in pots under a natural environmental condition. Theoretically, O. caudatum bulbs, which were grown in pots under a natural environmental condition. Theoretically, the expressions of OcUGlcAE3 in sterile bulbs and O. caudatum bulbs should be consistent. Actually, the expressions of OcUGlcAE3 in sterile bulbs and O. caudatum bulbs should be consistent. Actually, the the transcript level of OcUGlcAE3 in sterile bulbs was significantly higher than that in O. caudatum transcript level of OcUGlcAE3 in sterile bulbs was significantly higher than that in O. caudatum bulbs, bulbs, indicating environmental conditions had an effect on the expression of OcUGlcAE3 (Figure 9). indicating environmental conditions had an effect on the expression of OcUGlcAE3 (Figure 9). Also, Also, OcUGlcAE1 and OcUGlcAE2 were also mainly expressed in roots. The expression levels of OcUGlcAE1 and OcUGlcAE2 were also mainly expressed in roots. The expression levels of OcUGlcAE1 OcUGlcAE1 and OcUGlcAE2 were barely detected in bulbs, leaves and flowers (Figure 9). and OcUGlcAE2 were barely detected in bulbs, leaves and flowers (Figure 9). 2.8. Discussion There were at least four GalA-containing polysaccharides, which exhibited potent anticancer activity against Sarcoma 180 solid tumor, in O. caudatum [16]. UDP-GalA was reasonably inferred to be the sole donor for these polysaccharides biosynthesis. However, the genes responsible for UDP-GalA biosynthesis in O. caudatum had never been documented. In this research, a small UGlcAE family with three candidate members was isolated from O. caudatum for the first time. In vitro reaction verified the activity of OcUGlcAE3, interconverting UDP-GlcA and UDP-GalA. Previous experiments showed that these polysaccharides extracted from the whole plant [16], indicating the root

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of O. caudatum might also contain these anti-cancer polysaccharides. The high expression of OcUGlcAE3 in the root was Molecules 2016, 21, thus 1505 indicative of its involvement in the biosynthesis of GalA-containing polysaccharides 10 of 16 in roots. Of course, all of this need to be further verified through experiments.

Figure 9. 9. Expression Expressionprofiles profiles three OcUGlcAE genes in various O. caudatum tissues including Figure ofof thethe three OcUGlcAE genes in various O. caudatum tissues including sterile sterile bulbs, roots, bulbs, leaves and flowers by RT-qPCR. Relative transcript levels were standardised bulbs, roots, bulbs, leaves and flowers by RT-qPCR. Relative transcript levels were standardised to the to the constitute GAPDH expression. shown represent average ± (standard SD (standard deviation) constitute GAPDH gene gene expression. DataData shown represent the the average ± SD deviation) of of three independent experiments. three independent experiments.

2.8. Discussion Although there had been few reports of the application of UGlcAEs in microbial production of GalA-containing molecules present, the successful characterization of OcUGlcAE3 would no There were at least fouratGalA-containing polysaccharides, which exhibited potent anticancer doubt provide a more choice for gene parts used for GalA-containing molecules production in activity against Sarcoma 180 solid tumor, in O. caudatum [16]. UDP-GalA was reasonably inferred to be engineered cell factories in the future. So far, the plant UGlcAE genes were mainly isolated from the sole donor for these polysaccharides biosynthesis. However, the genes responsible for UDP-GalA Arabidopsis [3,5,14,15], mays [5]and Oryza sativa japonica [5]. Compared to these isoforms, biosynthesisthaliana in O. caudatum hadZea never been documented. In this research, a small UGlcAE family with the K m value for UDP-GlcA of OcUGlcAE3 was relatively low, indicating its higher affinity to the three candidate members was isolated from O. caudatum for the first time. In vitro reaction verified the substrate unlike AtUGlcAE1 Arabidopsis, a wider activity of UDP-GlcA. OcUGlcAE3,Moreover, interconverting UDP-GlcA and from UDP-GalA. PreviousOcUGlcAE3 experimentshad showed that temperature tolerance [3]. these polysaccharides extracted from the whole plant [16], indicating the root of O. caudatum might also contain these anti-cancer polysaccharides. The high expression of OcUGlcAE3 in the root was

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thus indicative of its involvement in the biosynthesis of GalA-containing polysaccharides in roots. Of course, all of this need to be further verified through experiments. Although there had been few reports of the application of UGlcAEs in microbial production of GalA-containing molecules at present, the successful characterization of OcUGlcAE3 would no doubt provide a more choice for gene parts used for GalA-containing molecules production in engineered cell factories in the future. So far, the plant UGlcAE genes were mainly isolated from Arabidopsis thaliana [3,5,14,15], Zea mays [5] and Oryza sativa japonica [5]. Compared to these isoforms, the Km value for UDP-GlcA of OcUGlcAE3 was relatively low, indicating its higher affinity to the substrate UDP-GlcA. Moreover, unlike AtUGlcAE1 from Arabidopsis, OcUGlcAE3 had a wider temperature tolerance [3]. In vitro functional identification of OcUGlcAE3 was carried out on the premise that the soluble recombinant OcUGlcAE3 should be yielded. It was expected that the active recombinant protein could not been detected when the full-length OcUGlcAE3 gene was expressed in E. coli due to the presence of the trans-membrane domains in its N-terminus. Truncated mutant of OcUGlcAE3 was thus generated by a deletion of a putative trans-membrane domain of 128 aa in the N-terminus. Unexpectedly, no soluble expression of OcUGlcAE3 was induced to express in E. coli. This result was inconsistent with previous reports, where the soluble truncated UGlcAE enzymes from other organisms were obtained [3,5]. The reason for this discrepancy may be the length of amino acids deleted from the N-terminus of UGlcAEs. As illustrated in the supporting Table S2, there were one or two trans-membrane domains in varied UGlcAEs. The first domain is generally located between 30–70 aa from the N-terminus, while the second motif lay in the domain between 85–140 aa (Table S2). A large majority of UGlcAE proteins have the first domain, while only a few UGlcAE isoforms have both domains (Table S2). In previous reports, the truncated UGlcAE enzymes were obtained by a deletion of the first trans-membrane domain [3,5]. The expression of these truncated UGlcAE genes generally leads to soluble UGlcAE proteins. On the contrary, when truncated OcUGlcAE3 with a deletion of two trans-membrane domains were introduced into E. coli for expression, no soluble recombinant OcUGlcAE protein was obtained. These data collectively revealed the length of amino acids removed from the N-terminal of UGlcAEs had effect on the soluble expression. Moreover, the length of amino acids removed from UGlcAE enzymes also affects the activities of UGlcAE proteins. The truncated UGlcAEs with a deletion of no more than 72 aa in previous reports exhibited 4-epimerase activity, catalyzing the interconversion of UDP-GlcA and UDP-GalA [3,5]. However, the truncated OcUGlcAE3 had no action on UDP-GlcA. The truncated OcUGlcAE3 removed a stretch of 128 aa from its N-terminus, where the GxxGxxG motif located (Figure S6). On the contrary, those UGlcAEs with a truncation of no more than 72 aa kept the presence of GxxGxxG motif, which was as illustrated in Figure S6. GxxGxxG motif is a conserved domain that performs the function of binding NAD+ . NAD+ is a cofactor which is required for UGlcAEs activity. The deletion of the conversed GxxGxxG motif in turn resulted in the activity loss of OcUGlcAE3. Together, these data indicated the N-terminus of UGlcAEs played important roles in their soluble expression and 4-epimerase activity. 3. Materials and Methods 3.1. Plasmids, Strains and Compounds Plasmids pEASY® -Blunt (TransGen Biotech Co. Ltd., Beijing, China) and pET-28a (+) (Novagen, Madison, WI, USA) were used for gene amplification and heterologous expression, respectively. Integration plasmid pPIC3.5K (Invitrogen, Carlsbad, CA, USA) was functional in Pichia strain GS115. E. coli strains Trans1-T1 and Transetta (DE3) were obtained from TransGen Co. Ltd. and were used as a bacterial host for recombinant plasmids amplification and enzymes expression, respectively. Pichia pastoris GS115 was used as a eukaryotic host for heterologous expression of OcGlcAE1~3. The detailed plasmids and strains used in this study are provided in Table S1.

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UDP-D-GalA was from CarboSource Services (University of Georgia, Athens, GA, USA). UDP-D-GlcA, NAD+ and NADH were obtained from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). All other chemicals used in this study were of analytical grade. 3.2. Bacterial and Yeast Culture Media E. coli strain was grown in Luria-Bertani medium (10 g/L Bacto-Tryptone, 5 g/L Bacto-yeast extract, 10 g/L NaCl) supplemented with appropriate antibiotics when required for selection. Yeast extract–peptone–dextrose (YPD) medium (2% (w/v) peptone, 1% (w/v) yeast extract, and 2% (w/v) dextrose) was used for yeast culture. Minimal dextrose (MD) medium (1.34% YNB, 4 × 10−5 % biotin, 2% dextrose, 1.5% agar) was applied in selection of yeast His+ transformants. The buffered minimal glycerol-complex (BMGY) medium (100 mM potassium phosphate buffer (pH 6.0), 2% peptone, 1% yeast extract, 1.34% yeast nitrogen base with ammonium sulphate but without amino acids, 1% glycerol, and 4 × 10−5 % biotin) was prepared for yeast protein induction. 3.3. Plant Material O. caudatum plants were maintained in our experimental pots or sterile conditions as previously described [17,18]. O. caudatum tissues were collected as described by Li et al. [18] and used as the starting materials for cDNA isolation and RT-qPCR analyses of OcUGlcAEs. 3.4. Unigenes Retrieval from O. caudatum RNA-Seq Data Transcriptome sequencing of O. caudatum was carried out previously [19,24]. The resulting massive unigenes were functionally annotated by Blast X against protein database deposited in GenBank library. Unigenes sharing highly similarity to UGlcAE were hence retrieved from RNA-Seq data of O. caudatum. 3.5. cDNA Isolation and Bioinformatics Analyses of OcUGlcAEs To verify the authenticity of the retrieved unigenes, cDNA amplification was performed using gene-specific primers by nested PCR. The resulting cDNA sequences were then sequenced for their identity and further analyzed by bioinformatics tools in details as introduced by Li et al. [18]. A phylogenetic tree was constructed using the Neighbor-joining method with the molecular evolutionary genetics analysis (MEGA) software, version 7.0 [25]. The reliability of the tree was measured by bootstrap analysis with 1000 replicates. The primers used in this study were listed in supporting material Table S3. 3.6. Bacterial Expression of OcUGlcAEs Three truncated OcUGlcAE genes were inserted into HindIII/EcoRI linearized pET-28a (+) to yield three recombinant plasmids by In-fusion technology. The primers used for plasmids construction were listed in Table S3. These pET-28a (+) derived plasmids were then transformed into Transetta (DE3) for heterologous expression. The resultant E. coli crude extract was applied to perform SDS-PAGE analyses as described previously [17–19]. The enzyme activities of these OcUGlcAE proteins were examined in a total volume of 100 µL containing 5 µL UDP-GlcA (10 mM), 5 µL NAD+ (10 mM) and 30 µL crude protein extract. The mixture was incubated at 30 ◦ C for 30 min. The reaction was then stopped by adding 25 µL chloroform. After vortex and removal of proteins by 0.45 µm membrane, twenty microliters supernatant was directly analyzed by HPLC. 3.7. Yeast Expression and Functional Characterization of OcUGlcAE1 Three full-length OcUGlcAE genes were ligated with BamH I and EcoR I digested pPIC3.5K to generate three expression plasmids, pPIC3.5KOcUGlcAE1, pPIC3.5KOcUGlcAE2 and pPIC3.5KOcUGlcAE3.

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The three plasmids were then digested by SacI and the resultant SacI-linearized recombinant plasmids were transformed into P. pastoris by electroporation as described by the manufacturer. Cells were spread on MD medium and incubated at 30 ◦ C for 48–72 h until colonies formed. Several colonies were picked and re-inoculated in YPD plates containing increasing geneticin at a final concentration of 0, 2 and 4 mg/mL. The YPD plates were incubated at 30 ◦ C for 2 to 3 days. The colonies resistant to 4 mg/mL geneticin were picked for further PCR confirmation using 5’AOX1 and 3’AOX1 primers (Table S3). The positive P. pastoris transformants were grown in 15-mL volumes (50 mL baffled shake flasks) of BMGY medium at 30 ◦ C with constant shaking (220 rpm) until an OD600 of 5–6 was obtained. The induction of OcUGlcAEs was performed for 120 h by daily supplementation of filter-sterilized 100% methanol at a final concentration of 1%. The yeast cells were then harvested by low speed centrifugation (1000 g, 10 min, 4 ◦ C) and washed twice with ddH2 O. All further steps were at 4 ◦ C or on ice. The resultant pellets were re-suspended in phosphate buffer (pH 7.0) and subjected to be disrupted by high pressure (1200 bar, 6–8 times).The obtained homogenate was centrifuged at 12,000× g for 30 min to remove the cell debris. Next, the resultant supernatant at this stage was ultra-centrifuged at 160,000× g for 1.5 h to sediment the microsomes. The supernatant and microsomal fractions were used as the biocatalyst for interconversion of UDP-GlcA and UDP-GalA, respectively. The assay was performed in a total volume of 100 µL containing 5 µL UDP-GlcA (10 mM), 5 µL NAD+ (10 mM) and 30 µL supernatant. The mixture was incubated at 30 ◦ C for 30 min. The reaction was then stopped by adding 25 µL chloroform. After vortex and removal of proteins by 0.45 µm membrane, the samples were analyzed by HPLC (high performance liquid chromatography) and LC-MS. HPLC was performed on a Hitachi Elite Lachrom HPLC system (Hitachi, Tokyo, Japan) equipped with a diode array detector and Dionex CarboPacTM PA10 carbohydrate column (4 × 250 mm, Thermo, Waltham, MA, USA). For generation of an analytical scale, the mobile phase consisted of ddH2 O (mobile phase A) and acetic acid-ammonium acetate buffer (700 mM, pH 5.2, mobile phase B). The concentration gradient of mobile phase B was programmed as follows: 0–20 min, 5%; 20–32 min, 45%; 32–33 min, 100%; 33–40 min, 5%. The flow rate of the mobile phase was 1 mL/min. Detector signal was monitored at a wavelength of 261 nm. The column temperature was kept ambient and injection volume was 20 µL. LC-MS was performed as described by Li et al. [18]. 3.8. Enzymatic Characterization of OcUGlcAE3 To obtain a comprehensive knowledge of OcUGlcAE3, the optimum pH and temperature evaluation and kinetic analyses of OcUGlcAE3 were performed as described previously with some modifications [17,18,26]. To determine the effect of pH on OcUGlcAE3, the supernatant from P. pastoris expressing OcUGlcAE3 was incubated with UDP-GlcA in sodium acetate-acetic acid buffer (pH 4) and phosphate buffered saline (PBS, pH 5–11) at 37 ◦ C for 1 h. For determination of optimal temperature, reactions were conducted for 1 h in 100 mM PBS buffer (pH 7.8) between 0 ◦ C and 60 ◦ C with a 5 ◦ C interval. These assays were carried out using UDP-GlcA as the substrate. The formation of UDP-GalA was monitored by HPLC. Each assay was performed in triplicate. Kinetic analysis of OcUGlcAE3 was conducted in 200 µL PBS buffer (100 mM, pH 7.8) containing 30 µL supernatant from P. pastoris expressing OcUGlcAE3 and varied concentrations of UDP-GlcA in the range of 0.156–10 mM. These assays were performed at 37 ◦ C for 20 min. Each assay was performed in triplicate. The product formation was monitored by HPLC at 259 nm. The rates of product formation were thus determined as a function of substrate concentrations. The kinetic values were thus calculated by double reciprocal plot (Lineweaver-Burk plot), which was constructed by Graphpad Prism 5.01 software. 3.9. Expression Analyses of OcUGlcAEs in O. caudatum Expression analyses of OcUGlcAEs in varied tissues including roots, bulbs, leaves, flowers and sterile bulbs were performed using RT-qPCR technology described in our recent publications [17,18].

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GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal control to normalize the expression level of OcUGlcAE genes. The gene-specific primers used for RT-qPCR assay were designed according to Primer Express 3.0 software (Applied Biosystems, Waltham, MA, USA) and summarized in Table S3. 4. Conclusions UDP-GalA is an important donor for GalA-containing polysaccharides in O. caudatum. However, the genes responsible for the UDP-GalA biosynthesis had not been documented. In this investigation, a small UGlcAE family with three members was isolated from O. caudatum based on RNA-Seq data for the first time. OcUGlcAE3 was then identified to be a bona fide gene by in vitro enzymatic action. RT-qPCR was thus applied to analyze the expression profile of OcUGlAE3. Data showed that the expression of OcUGlcAE3 was root-specific and OcUGlcAE3 was hence deemed to be involved in the biosynthesis of root-specific GalA-containing polysaccharides. Moreover, OcUGlcAE3 was proposed to be environmentally induced. Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/ 11/1505/s1. Acknowledgments: This work was supported by Independent Subject of Key Project of State Key Laboratory of Bioactive Substance and Function of Natural Medicines (GTZA201404) and the Fundamental Research Funds for the Central Universities of Chinese Academy of Medical Sciences & Peking Union Medical College (2016ZX350001). Author Contributions: S.Y. and Y.-J.S. performed the experiments; M.L. performed HPLC analyses; L.-N.L. isolated the total RNA of the plants; J.-Q.K. conceived and designed the experiments, analyzed the data and wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations BMGY GalA GAPDH HPLC MD ORF PBS RT-qPCR SDR trun-OcUGlcAEs UDP-Ara UDP-Gal UDP-GalA UDP-Glc UDP-GlcA UDP-Xyl UGlcAE UGE UTR UXE YPD

buffered minimal glycerol-complex medium D -galacturonic acid Glyceraldehyde-3-phosphate dehydrogenase High performance liquid chromatography Minimal dextrose open reading frame Phosphate buffered saline real-time quantitative PCR short-chain dehydrogenase/reductase Truncated OcUGlcAE proteins UDP-L-arabinose UDP-D-galactose UDP-D-galacturonic acid UDP-D-glucose UDP-D-glucuronic acid UDP-D-xylose UDP-D-glucuronic acid 4-epimerase UDP-D-glucose 4-epimerase untranslated region UDP-D-xylose 4-epimerase yeast extract–peptone–dextrose medium

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Sample Availability: OcUGlcAE3 gene and corresponding plasmids are available from the authors. © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).