Effect of flexibility and positive charge of the Cterminal domain on the ...

RESEARCH LETTER

Effect of flexibility and positive charge of the C-terminal domain on the activator P14K function for nitrile hydratase in Pseudomonas putida Yi Liu1, Wenjing Cui1, Zhongmei Liu1, Youtian Cui1, Yuanyuan Xia1, Michihiko Kobayashi2 & Zhemin Zhou1 1

Key Laboratory of Industrial Biotechnology, School of Biotechnology, Jiangnan University, Wuxi, China; and 2Institute of Applied Biochemistry, The University of Tsukuba, Tsukuba, Ibaraki, Japan

Correspondence: Zhemin Zhou, Key Laboratory of Industrial Biotechnology, School of Biotechnology, Jiangnan University, Wuxi 214122, China. Tel.: +86 510 85325210; fax: +86 510 85197551; e-mail: [email protected] Received 15 December 2013; accepted 31 December 2013. Final version published online 30 January 2014. DOI: 10.1111/1574-6968.12376

Abstract A self-subunit swapping chaperone is crucial for cobalt incorporation into nitrile hydratase. However, further information about its structural features is not available. The flexibility and positive charge of the C-terminal domain of the selfsubunit swapping chaperone (P14K) of nitrile hydratase from Pseudomonas putida NRRL-18668 play an important role in cobalt incorporation. C-terminal domain truncation, alternation of C-terminal domain flexibility through mutant P14K(G86I), and elimination of the positive charge in the C-terminal domain sharply affected nitrile hydratase cobalt content and activity. The flexible, positively charged C-terminal domain most likely carries out an external action that allows a cobalt-free nitrile hydratase to overcome an energetic barrier, resulting in a cobalt-containing nitrile hydratase.

MICROBIOLOGY LETTERS

Editor: Simon Silver Keywords protein expression; nitrile hydratase; enzyme activation; cobalt incorporation.

Introduction Nitrile hydratase (NHase; EC4.2.1.84), which is composed of a- and b-subunits, contains either a nonheme iron (Fe-NHase; Greene & Richards, 2006) or a noncorrin cobalt ion (Co-NHase; Kobayashi & Shimizu, 1999) in the active center and catalyzes the hydration of a nitrile (RCN) to the corresponding amide (RCONH2). This reaction is followed by consecutive reactions, amide ? acid ? acylCoA, which are catalyzed by amidase (Kobayashi et al., 1997) and acyl-CoA synthetase (Noguchi et al., 2003), respectively. The metal ions in both the Co-NHase and Fe-NHase are located in the a-subunits, which share a characteristic metal-binding motif CXLC(SO2H)SC(SOH) that contains two modified cysteine residues: cysteinesulfinic acid (aCys-SO2H) and cysteine-sulfenic acid (aCys-SOH; Murakami et al., 2000; Noguchi et al., 2003). The apoenzyme is likely to be unmodified (Miyanaga et al., 2004) and a related enzyme is the thiocyanate hydrolase (Katayama et al., 2006). The noncorrinoid cobalt has ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

received increasing interest in bioinorganic chemistry and in biotechnology. Its availability and remarkable chemical versatility make it an invaluable catalyst in the chemical industry (Mitra et al., 2008). The trafficking of metal ions into NHases is mediated by activator proteins (Okamoto & Eltis, 2011). Fe-NHases in Rhodococcus, Pseudomonas chlororaphis, and Rhodococcus require activators for functional expression (Nishiyama et al., 1991; Hashimoto et al., 1994; Nojiri et al., 1999). A proposed metal-binding motif, CXCC, in the NHase activator of Rhodococcus sp. N-771 has been identified, and the activators for Fe-type NHases have been shown to act as metallochaperones (Lu et al., 2003). The gene organizations are quite different among the Co-NHases, and their cobalt incorporation mechanisms are also different. One type of Co-NHases has the gene order , such as the NHases from Rhodococcus rhodochrous J1 (Zhou et al., 2008). The second type has the gene order , FEMS Microbiol Lett 352 (2014) 38–44

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Flexibility and positive charge of P14K

such as the NHase from Rhodococcus jostii RHA1 (Okamoto et al., 2010). The third type has the gene order , such as the NHase from Pseudomonas putida NRRL-18668 (Liu et al., 2013). Although the activator of the NHase from R. jostii strain acts as a metallochaperone for cobalt incorporation (Okamoto et al., 2010), cobalt incorporation into the NHases from R. rhodochrous J1 and P. putida NRRL-18668 is dependent on a novel mode of post-translational maturation called self-subunit swapping. The activator protein exists as a complex with the a-subunit of NHase, and cobalt incorporation involves swapping the cobalt-free a-subunit of the cobalt-free NHase with the cobalt-containing a-subunit of the complex (Zhou et al., 2008; Liu et al., 2012). Self-subunit swapping is quite different from the known general mechanisms of metallocenter biosynthesis thus far and reveals the unexpected behavior of a protein in a protein complex (Zhou et al., 2008). Various other Co-NHases and an NHase family enzyme, thiocyanate hydrolase, most likely also maturate through selfsubunit swapping (Zhou et al., 2010). The self-subunit swapping chaperones that exhibit a surprising protein function are recognized as self-subunit swapping chaperones in contrast with other metallochaperones involved in metallocenter biosynthesis and molecular chaperones in protein folding (Zhou et al., 2008). In addition, selfsubunit swapping chaperones also are crucial for the post-translational cysteine oxidation in the NHase active center (Zhou et al., 2008). However, there is no further information on the structural features of self-subunit swapping chaperones, and the relationship between their structural features and functions remains unclear. In the present study, we investigated the relationship between the structural features and the function of P14K in P. putida NRRL-18668. We found that the flexibility and positive charge of the C-terminal domain (C-domain) of P14K underlay its function of cobalt incorporation during NHase activation.

Materials and methods Bacterial strains and plasmids

P14K is barely detectable in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). However, N-terminal addition of a strep tag, an artificial peptide used for the purification and detection of recombinant fusion proteins, can enhance the expression of P14K (Wu et al., 1997; Liu et al., 2013). Therefore, pET-AB-strepP (Liu et al., 2013), a plasmid containing the a- and b-subunit genes (AB) and a strep-tagged P14K gene, was used for the expression of FEMS Microbiol Lett 352 (2014) 38–44

the NHase and P14K, and used as a template for mutagenesis or truncation. Escherichia coli JM109 was the host for the cloning work. Escherichia coli BL21 (DE3) was used as the host strain for gene expression. Corresponding primers for the mutants were shown in Supporting Information Table S1. Expression and purification of enzymes

Escherichia coli BL21 (DE3) transformants containing the recombinant plasmids were grown at 37 °C in TB medium (12 g L 1 tryptone, 24 g L 1 yeast extract, 4 g L 1 glycerol, 17 mM KH2PO4, and 72 mM K2HPO4) contain(50 mg L 1) and kanamycin ing CoCl2.6H2O 1 (50 lg mL ) until the A600 nm reached 0.8. Isopropyl b-D-thiogalactopyranoside was added to a final concentration of 0.4 mM, and then, the cells were incubated at 24 °C for 16 h. All purification steps were performed at 4 °C, and the procedures were conducted with an AKTA purifier (GE Healthcare UK Ltd.). Potassium phosphate buffer (10 mM, pH 7.5) containing 0.5 mM dithiothreitol (DTT) was used in the purification steps. NHase and P14K were purified as described previously (Liu et al., 2013). Enzyme assay

The NHase activity was assayed in a reaction mixture comprising 10 mM potassium phosphate (pH 7.5), 20 mM 3-cyanopyridine as a substrate, and 0.1 lg enzyme in a total volume of 500 lL. The reaction was carried out at 20 °C for 20 min and stopped by the addition of 0.5 mL acetonitrile. One unit of NHase activity was defined as the amount of enzyme that catalyzed the release of 1 lmol of nicotinamide per min at 20 °C (Zhou et al., 2008). Analytical methods

The UV-Visible spectra were obtained with a U-0080D spectrophotometer (Hitachi, Tokyo, Japan) at room temperature. The enzymes were dialyzed against 10 mM potassium phosphate (pH 7.5), and 1.0 mg mL 1 samples were prepared. Homology modeling of P14K

The MODELLER 9.7 (Eswar et al., 2007) software was used for the structure prediction. Stereochemical analysis of the structures was performed using the PROCHECK software (http://nihserver.mbi.ucla.edu/SAVS/), and the final models that displayed good geometry (with < 1% ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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of residues in the disallowed region) were used in this study. Quantum chemical calculations of the transition state

All calculations were carried out using the MOPAC2012 program. The energetic profile of the reaction barrier was mapped out by carrying out conventional self-consistent field calculations (http://OpenMOPAC.net).

Results and discussion The simulation of structure fluctuations of P14K

To investigate the structural features of P14K, molecular dynamic simulations were carried out. P14K and other self-subunit swapping chaperones exhibit meaningful sequence similarity to the NHase b-subunits (Zhou et al., 2010). Therefore, P14K was modeled using the N-terminal domain of b-subunit of NHase as the template as reported previously (Cameron et al., 2005). The average RMSF (root-mean-square fluctuation) per residue for the backbone atoms was calculated using a fast simulation of protein structure fluctuations online server CABS-flex (http://biocomp.chem.uw.edu.pl/CABSflex/). On the basis of the mean RMSF, residues L85-A144 of the C-domain are considered to be flexible (Fig. 1a).

The C-domain of P14K is important for its function

To investigate the effect of C-domain on the function of P14K, a mutant gene AB-strepP(DC) was designed, in which the residues L85-A144 (60 amino acids) of the C-domain of P14K were truncated, and constructed the pET-AB-strepP(DC) plasmid. The transformant harboring pET-AB-strepP(DC) was used for NHase expression (Fig. 1b). The NHase encoded by the gene AB-strepP(DC) was purified (Fig. 1c) and used to compare with the wild-type NHase encoded by gene AB-strepP. The activity of the NHase encoded by the gene AB-strepP(DC) was only 3% that of the wild-type NHase (Table 1). This result demonstrated that the C-domain plays an important role in the function of P14K. The effect of the flexibility of the C-domain on P14K function

To investigate how the C-domain affects the function of P14K, we aligned several self-subunit swapping chaperones (Fig. 2). Gly86 conserved in all of these self-subunit swapping chaperones. Further, we analyzed the modeled structure of P14K, the C-domain appears to be a long loop despite containing two short a-helixes, and there are three Glys (Gly86, Gly90, and Gly91) at its start point (Fig. 3a). According to the general relationships between the Gly, loop structure, and flexibility (Epand et al., 1986;

(a)

(b)

(c)

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Fig. 1. Flexibility analysis of P14K and SDSPAGE analysis of NHases and P14K-containing complexes [a(P14K)2]. (a) The average calculated RMSF (root-mean-square fluctuation) per residue for the backbone atoms. The dash ellipse surrounds the C-domain. (b) SDS-PAGE analysis of lane (1) MW markers and the cell extracts of the transformants carrying (2) pET-AB-strepP, (3) pET-AB-strepP(DC), (4) pET-AB-strepP(C-G86I), (5) pET-AB-strepP(C-sixH), (6) pET-AB-strepP (C-R96A), (7) pET-AB-strepP(C-12positive). (c) SDS-PAGE analysis of lane (1) MW markers and purified NHases from the transformants carrying (2) pET-AB-strepP, (3) pET-AB-strepP (DC), (4) pET-AB-strepP(C-G86I), (5) pETAB-strepP(C-sixH), (6) pET-AB-strepP(C-R96A), (7) pET-AB-strepP(C-12positive), and the a (P14K)2 from the transformants carrying (8) pET-AB-strepP, (9) pET-AB-strepP(C-G86I), (10) pET-AB-strepP(C-12positive).

FEMS Microbiol Lett 352 (2014) 38–44

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Nilmeier et al., 2011), we considered that the C-domain possesses certain flexibility. Protein function is intimately linked to protein flexibility, and any interaction between a protein and another molecule requires the protein to be able to change its conformation. Proteins rely on flexibility to respond to

Table 1. Activity of the purified NHases co-expressed with the P14K mutants in Escherichia coli Plasmid (NHase)

NHase activity, U mg 1 protein

pET-AB-strepP *pET-AB-strepP(DC) *pET-AB-strepP(C-G86I) *pET-AB-strepP(C-sixH) *pET-AB-strepP(C-R96A) *pET-AB-strepP(C-K101A) *pET-AB-strepP(C-K127A) *pET-AB-strepP(C-K129A) *pET-AB-strepP(C-R134A) *pET-AB-strepP(C-K138A) *pET-AB-strepP(C-12positive)

439 12 42 310 422 420 406 410 411 416 20

3.6 1.2 6.0 5.3 1.6 1.4 3.6 4.3 7.1 3.7 4.9

The values represent the means SD for at least triplicate independent experiments. pET-AB-strepP contains the a- and b-subunit genes (AB) and a strep-tagged P14K gene (strepP). pET-AB-strepP(DC) contains AB genes and a mutant strep-tagged P14K gene (strepP(DC)) in which the residues L85-A144 (60 amino acids) were truncated. pETAB-strepP(C-sixH) contains AB genes and a mutant strep-tagged P14K gene (strepP(C-sixH)) in which six histidines (H104, H109, H111, H117, H128, and H130) were substituted with neutral Alanines. pETAB-strepP(C-12positive) contains AB genes and a mutant strep-tagged P14K gene (strepP(C-12positive)) in which 12 positive amino acids (H104, H109, H111, H117, H128, H130, R96, K101, K127, K129, R134, and K138) were substituted with neutral alanines. *The character C means the C-domain of P14K.

environmental changes; a perturbation that changes the flexibility of a protein may potentially interfere with its function (Teilum et al., 2011). We modeled a structure of mutated P14K, in which Gly86 was substituted by Ile; a large side-chain amino acid such as Ile would likely be an obstacle for the conformational flexibility of the C-domain. The average RMSF per residue was calculated using the online server CABS-flex (Fig. 1a). Compared to the wild-type P14K, the flexibility of the C-domain of P14K(G86I) was decreased, while that of the rest of the protein was similar. Subsequently, we designed the mutant gene AB-strepP(C-G86I) and constructed the plasmid pET-AB-strepP(C-G86I). The transformant harboring pET-AB-strepP(C-G86I) was then used for NHase expression (Fig. 1b). The NHase encoded by the gene AB-strepP (C-G86I) was purified (Fig. 1c) and used to compare with the wild-type NHase. The activity of the NHase encoded by gene AB-strepP(C-G86I) was only 10% that of the wild-type NHase (Table 1). These findings demonstrated that the flexibility of the C-domain might be an important factor for P14K function. The positively charged amino acids of the C-domain might be crucial for P14K function

NHase is a metalloprotein, and P14K is essential for cobalt incorporation into NHase (Liu et al., 2012). In addition, the C-domain of P14K plays an important role in P14K function (Table 1). These findings indicate that there may be some functional amino acid residues related to cobalt binding in this domain. Six histidine residues (H104, H109, H111, H117, H128, and H130) in the C-domain (Fig. 2) were proposed to be involved in cobalt binding because histidine is considered to have a high

Fig. 2. Protein sequence alignment of selfsubunit swapping chaperones. PP P14K, RAPc8 P14K, J1 E, and J1 G indicate the selfsubunit swapping chaperones of NHases in Pseudomonas putida NRRL-18668, Bacillus RAPc8, and Rhodococcus rhodochrous J1 (the two self-subunit swapping chaperones for low and high molecular mass NHases are represented by E and G), respectively. Closed points indicate Gly86 and Arg96, and asterisks indicate the 12 positively charged amino acids (H104, H109, H111, H117, H128, H130, R96, K101, K127, K129, R134, and K138), respectively, in the C-domain of P14K of P. putida NRRL-18668.



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(a)

(b)

Fig. 3. The structure of P14K obtained by homology modeling and the surface electrostatic potentials prediction. (a) The a-helical regions of the 60 amino acids in C-domain are shown as cylinder. The residues displaying side chain represent Gly86, Gly90, and Gly91, respectively. The dashed curve represents the surface of the C-domain of P14K. (b) The surface electrostatic potentials of P14K and the cobalt-free a-subunit. I, the surface electrostatic potentials of P14K; II, the surface electrostatic potentials of the cobalt-free a-subunit. The positive charges and negative charges are shown in blue and red, respectively.

affinity for metals. We designed a gene AB-strepP(C-sixH) in which the six His residues were substituted with Ala and constructed the plasmid pET-AB-strepP(C-sixH). The transformant harboring pET-AB-strepP(C-sixH) was used for the mutant NHase expression (Fig. 1b), and the NHase encoded by gene AB-strepP(C-sixH) was purified (Fig. 1c) and used to compare with the wild-type NHase. 70% activity of the wild-type NHase still remained in this mutant (Table 1), indicating that these His residues only partially participate in P14K function. In addition, residue Arg96, which is conserved among the self-subunit swapping chaperones (Fig. 2), was also changed to investigate any effect on P14K function. The purified mutant NHase encoded by AB-strepP(C-R96A) exhibited the same level of activity as the wild-type NHase (Table 1), showing that the Arg96 residue does not affect P14K function. As there are no other conservative amino acids in the C-domain besides above referred Gly86 and Arg96 (Fig. 2), molecular electrostatic potentials (MEP) were used for further analysis of the C-domain. P14K has been confirmed to form a complex with the a-subunit of ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

NHase (Liu et al., 2012). Therefore, we modeled the MEP of both P14K and the a-subunit using the Adaptive Poisson-Boltzmann Solver (APBS; Baker et al., 2001) implemented in PyMOL (Fig. 3b). The flexible C-domain forms a positively charged region on the surface of P14K, similar to a positively charged stick, while the active center in the a-subunit forms a negatively charged region, indicating that the flexible C-domain might effectively access the a-subunit via the molecular electrostatic pulling forces between the positively charged region in C-domain and the negatively charged active center in the a-subunit. Following to this speculation, other five positively charged amino acids (K101, K127, K129, R134, and K138) in the C-domain (Fig. 2) were substituted by Ala, respectively, and each mutant NHase was purified. Each mutation had little influence on NHase activity (Table 1). However, when all the 12 positive amino acids (H104, H109, H111, H117, H128, H130, R96, K101, K127, K129, R134, and K138) in the C-domain (Fig. 2) were substituted with neutral Ala, the activity of this purified mutant NHase (encoded by AB-strepP(C-12positive)) decreased to c. 5% that of the wild-type NHase (Table 1). These findings demonstrated that the total positively charged environment of C-domain plays an important role in P14K function. The flexibility and positive charge of the C-domain related to cobalt incorporation

P14K forms a complex a(P14K)2 with the a-subunit of the NHase, and the incorporation of cobalt into the NHase of P. putida was confirmed to be dependent on the a-subunit substitution between the cobalt-containing a(P14K)2 and the cobalt-free NHase (self-subunit swapping; Liu et al., 2012). P14K acts not only as a chaperone for self-subunit swapping but also as a metallochaperone that is crucial for cobalt insertion into the a-subunit (Zhou et al., 2009; Liu et al., 2012). The low activity of the mutant NHases [encoded by AB-strepP(C-G86I) and AB-strepP(C-12positive)] may be caused by low cobalt content in the a-subunit of a(P14K)2 and then affects the cobalt content in NHase. Subsequently, the mutant NHases and the mutant a(P14K)2 [encoded by AB-strepP (C-G86I) and AB-strepP(C-12positive), respectively] were purified (Fig. 1c) and used to compare with the wild-type NHase and the wild-type a(P14K)2. The absorption in the 300–350 nm region of Co-NHase reflects the S->Co3+ charge transfer (Zhou et al., 2009), and an extra shoulder in the 300–350 nm is found in cobalt-containing a (P14K)2 and NHase but not in cobalt-free a(P14K)2 and NHase (Liu et al., 2012). The extra shoulder in the 300– 350 nm region was only observed in the wild-type a (P14K)2 and NHase, but not in the two mutant enzymes FEMS Microbiol Lett 352 (2014) 38–44

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(a)

(b)

(c)

Fig. 4. UV-Visible absorption spectra of the purified NHase and a (P14K)2 and quantum chemical and energy calculation. (a) UV-Visible absorption of the purified a(P14K)2 expressed from the transformants harboring pET-AB-strepP, pET-AB-strepP(C-G86I), and pET-AB-strepP (C-12positive), respectively. (b) UV-Visible absorption of the purified NHase expressed from the transformants harboring pET-AB-strepP, pET-AB-strepP(C-G86I), and pET-AB-strepP(C-12positive), respectively. (c) The crystal structure of the NHase in Pseudonocardia thermophila (PDB: 1UGQ, 95% amino acid identity) was used as the template for the homology modeling of immature NHase. The crystal structure of mature NHase is known (PDB: 3QXE). S1, S2, S3, N1, and N2 indicate the S and N atoms corresponding to cobalt binding in the active center. The distances and dihedrals among the cobalt-linked atoms of the active center in the immature NHase are S1-N1 3.617 A, S2-N2 5.033 A, and S1-N2-N1-S3 57.9°, respectively, while those of the mature NHase are S1-N1 4.075 A, S2-N2 4.358 A and S1-N2-N1-S3 58.7°. TS indicates the transition state of NHase during cobalt incorporation. The numbers 16851, 16648, 16918, and 203 kcal mol 1 indicate the calculated total energy of the immature, transition state, and mature NHase and the energy barrier, respectively.

(Fig. 4a and b), indicating that the cobalt is not successfully incorporated into the two mutant a(P14K)2 and the corresponding NHases. In addition, the process of cobalt binding was simulated through quantum chemical calculations implemented using MOPAC2012. As the differences between the conformations of the immature and mature NHases in the cobalt-linked atoms of the active centers are insignificant, with < 0.7 A in distance and 0.8° in dihedral (Fig. 4c), it seems that NHase has the potential ability for cobalt incorporation itself. However, a high


calculated energetic barrier 203 kcal mol 1 was observed among the immature, transition state, and mature NHases (Fig. 4c). This finding indicates that the immature NHase cannot maturate on its own. Therefore, an external action must occur for the immature NHase to overcome the energetic barrier and result in a mature NHase. The C-domain of P14K most likely performs this external action because the high flexibility and positive charge permit the C-domain to associate with the active center of the a-subunit. As a result, the energetic barrier is overcome, resulting in the cobalt-containing mature NHase. These findings suggest that the flexible, positively charged C-domain of P14K might play an important role in cobalt incorporation into NHase. Self-subunit swapping chaperones are essential for cobalt incorporation into NHase. However, the direct experimental evidence for the mechanism of cobalt incorporation is limited. Further study of the disordered region of P14K would be useful for investigating the function of the self-subunit swapping chaperones involved in NHase biosynthesis.

Acknowledgements This work was supported by the National Natural Science Foundation of China (31070711), the Doctoral Scientific Research Fund Project of Jiangnan University of China (JUDCF10011), the General University Doctor Research, and the Innovation Program of Jiangsu Province of China (CXZZ11_0475).

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Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. Oligonucleotide primers used in this study.
