ysis mechanism (Vernon and Banks 1963) involving car- boxyl groups at the active ... and Asp (20) of T4 lysozyme (Weaver and Matthews. 1987) were aligned ...
Mol Gen Genet (1990) 222:278-283 © Springer-Verlag 1990
Structure of the beta-l,3-1,4-glucanase gene of B a c i l l u s m a c e r a n s : Homologies to other beta-glucanases Rainer Borriss I *, Knut Buettner 2, and Pekka Maentsaelae 3 1 Zentralinstitut ffir Genetik und Kulturpflanzenforschung der Akademie der Wissenschaften der DDR, DDR-4325 Gatersleben, German Democratic Republic 2 Bereich Mikrobiologie, Sektion Biologic, E.-M.-Arndt Universit/it Greifswald, DDR-2200 Greifswald, German Democratic Republic 3 Department of Biochemistry, University of Turku, SF-20500 Turku, Finland ReceivedJanuary 16, 1990 Summary. The nucleotide sequence of an 852 base pair (bp) D N A fragment containing the entire gene coding for thermostable beta-l,3-1,4-glucanase of Bacillus macerans has been determined. The b g l M gene comprises an open reading frame (ORF) of 711 bp (237 codons) starting with ATG at position 93 and extending to the translational stop codon TAA at position 804. The deduced amino acid sequence of the mature protein shows 70% homology to published sequences of mesophilic beta-l,3-1,4-glucanases from B. subtilis and B. amyloliquefaciens. The sequence coding for mature beta-glucanase is preceded by a putative signal peptide of 25 amino acid residues, and a sequence resembling a ribosomebinding site (GGAGG) before the initiation codon. By contrast with the processed protein, the N-terminal amino acid sequence constituting the putative leader peptide bears no or only weak homology to signal peptides of mesophilic Bacillus endo-beta-glucanases. The B. macerans signal peptide appears to be functional in exporting the enzyme to the periplasm in E. coll. More than 50% of the whole glucanase activity was localized in the periplasmic space and in the supernatant. Whereas homology to endo-l,4-beta-glucanases is completely lacking, a weak amino acid homology between the sequence surrounding the active site of phage T4 lysozyme and a sequence spanning residues 126 through 161 orB. macerans endo-beta-glucanase could be identified. Key words: Endo-l,3-1,4-beta-glucanase - D N A sequence - Bacillus macerans - Active site - T4 lysozyme
ses only beta-l,4- linkages adjacent to beta-l,3- linkages in mixed linkage beta-glucans such as lichenan or barley beta-glucan, but is not able to split beta-l,4- linkages in carboxymethylcellulose. Products of hydrolysis are cellobiosyltriose and cellotriosyltetraose (Anderson and Stone 1975). Although endo-beta-l,4-glucanases are widely distributed in various kinds of organisms, lichenase is known to be produced only by plants and certain bacteria, especially by representatives of the genus Bacillus (Borriss et al. 1980). Genes coding for that group of enzymes have been cloned from Bacillus subtilis (Cantwell and McConnell 1983), B. amyloliquefaciens (Borriss et al. 1985), B. macerans (Borriss et al. 1988), Bacterioides succinogenes (Erfle et al. 1988) and germinating barley (Fincher et al. 1986). Although amino acid sequences of mesophilic Bacillus spp. exhibit 90% similarity (Hofemeister et al. 1986), no apparent homology was detected when the Bacillus lichenases were compared with bacterial endo-beta-l,4glucanases or with barley endo-beta-l,3-1,4-glucanase (Fincher et al. 1986). Endo-beta-l,3-1,4-glucanase from B. macerans is unique in its thermostability and has been found very useful for decreasing the viscosity of brewing mashes (Borriss and Schroeder 1981). Here we report extensive similarities between the mature forms of thermophilic B. macerans beta-glucanase and cloned mesophilic enzymes. The significance is discussed of the limited homology between a region conserved in all known Bacillus endo-beta-l,3-1,4-glucanases and the N-terminal active site region of phage T4 lysozyme.
Introduction Materials and methods Lichenase (1,3-1,4-beta-D-glucan-4-glucanohydrolase, E.C.3.2A.73) is an endo-beta-glucanase which hydrolyPresent address and offprint requests to: R. Borriss, Bereich Mikro-
biologic, Sektion Nahrungsgiiterwirtschaft, Humboldt=Universit/it Berlin, Warschauer Strasse 43/44, DDR-1017 Berlin
Bacterial strains, phages and plasmids. Plasmid pUCI 9/ 34 (chromosomal 0.85 kb D N A fragment from B. macerans cloned into pUC19 vector; Borriss et al. 1988)
was used for subcloning the beta-glucanase gene into M13 phage vectors mpl8 and mpl9 (Yanisch-Perron
279 et al. 1985). The host strains for transfection of phage DNA and transformation with plasmid DNA were Escherichia coli JM101 (Yanisch-Perron et al. 1985) and DH5cq a derivative of DH1 (Hanahan 1985). Media and growth conditions were as described previously (Borriss et al. 1988).
Enzymes and chemicals. Radioactive nucleotides were purchased from New England Nuclear. Restriction enzymes and T4 ligase were from Boehringer Mannheim; Klenow large fragment was supplied by Pharmacia. Lichenan was prepared from Iceland Moss (Cetraria islandica) as described by Borriss (1981). Transfection and transformation. The procedure for transfection of competent JM101 was according to the standard protocol given in the M13 Cloning and Sequencing Handbook (Amersham International). Transformation of E. coli DH5~ with plasmid DNA was performed according to a standard protocol (Maniatis et al. 1982). DNA purification. Plasmid DNA was prepared according to Maniatis et al. (1982). Single-stranded template preparation of M13 phages was done as described in the M13 Cloning and Sequencing Handbook (Amersham). Nucleotide sequencing. The dideoxy chain-termination sequencing procedure of Sanger et al. (1977) was employed as described previously (Hofemeister et al. 1986). The cloning strategy is outlined in Fig. 1. Other DNA methods not described above were carried out using standard techniques (Maniatis et al. 1982). Cell fractionation, enzyme and protein assays. Extracellular, periplasmic and cellular fractions were isolated as EcoBV
p19/A
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described by Cornelis et al. (1982). Activity detected in culture filtrates of E. coli was considered to represent extracellular beta-glucanase. The periplasmic beta-glucanase was the activity found in the supernatant following osmotic shock with cold water. The cellular fraction containing cytoplasmic and membrane-bound activities was obtained by sonication of washed cell suspensions. Beta-glucanase activity was measured colorimetrically using lichenan as substrate (Borriss 1981). One unit of enzyme released 1 gmol of glucose equivalents per rain. Protein concentration was determined by the dye binding method according to Bradford (1976). Beta-lactamase activity was assayed according to Perret (1954); glucose-6-phosphate-dehydrogenase was determined according to Ohmann et al. (1969).
Enzyme purification and amino acid sequencing. Beta-glucanase enzymes from different cell fractions were purified to homogeneity as described previously (Borriss et al. 1988). N-terminal amino acid sequence analysis was performed using a liquid phase sequenator (Beckman Instruments, Model 890 C). Results and discussion
Nucleotide sequence and expression in E. coli The nucleotide sequence of the 850 bp fragment (Fig. 1) containing the bglM gene was determined. We found one open reading frame (ORF), which starts from the ATG at position 93 and ends in TAA at position 804. The ORF encodes a polypeptide of 237 amino acid residues (Fig. 2). Previous work has shown that the bglM gene cloned into pUC vectors is well expressed in E. coli cells (Borriss et al. 1988). To confirm the putative start of translation we deleted DNA from the 5' end by cutting with restriction enyzmes DraI and BstEII (Figs. 1 and 2). The truncated fragments recloned in pUC19 started either 22nucleotides (nt) upstream (DraI) or 25 nt downstream (BstEII) from the putative start codon of the bgIM gene. By using site directed mutagenesis we introduced an artificial PvuII site at position 85, 7 nucleotides upstream from ATG and also recloned the deleted fragment into pUC19. As expected, betaglucanase activity was expressed from the DraI- and the PvuII fragments, but not from the BstEII fragment, confirming that the ATG at position 93 is the translation start site of the bglM gene. In addition, this experiment shows that expression of the blM gene in E. coli takes place in the absence of its own promoter sequence, using one of the relativly weak promoters located upstream of the cloned gene in the vector pUC19 plasmid. Attempts to clone the EcoRI-HindIII fragment containing the bgIM gene into pUC18 close to the strong lae promoter failed. All transformants obtained were unstable, possibly due to the high levels of expression of bglM gene product. Study of codon usage of the bglM gene reveals that UCG(Ser), CCC(Pro), ACU(Thr), CAC(His), CGC(Arg), CGG(Arg), AGA(Arg) and AGG(Arg) co-
280
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Fig. 2. Nucleotide sequence of the cloned glucanase gene and deduced amino acid sequence of the endo-beta-glucanase. The coding strand (5' to 3') is shown. Possible promoter regions P-35 and P-10 and RBS are overlined. The deduced amino acid sequence is shown in single letter code. Amino acids forming the putative signal peptide are numbered - 2 5 to - 1 . The signal peptide cleavage site ( A l a - 1 / G l y + i) has been determined by N-terminal sequencing of the mature protein (see text). Important restriction
sites are underlined in the nucleotide sequence. Non-coding sequences are indicated by small letters. The promotorless V6 flagment starts at the DraI restriction site at position 81, 12 nucleotides upstream from ATG. The sequence around the putative ribosome binding site was changed at two positions (GGAGG to GC*AGC*) by oligo mutagenesis in order to create an artificial PvuII site from (Kramer et al. 1984)
dons are not used. In the 5' flanking region of the bglM gene, sequences resembling the consensus regions of Bacillus o-43 promoters (Moran et al./982) have been identified (Fig. 2). The putative promoter spans positions 4 4 4 9 ( - 3 5 region) and 68-73 ( - 1 0 region) up to 20 nt upstream from the putative transcription start site. A possible ribosome binding site (SD) GGAGG sequence, complementary to the 3' end of 16S rRNA of B. subtilis (McLaughlin et al. 1981) precedes the bglM gene at nucleotides 82-86.
cantly contribute to the whole population of beta-glucanase molecules. By contrast with the mature proteins, no obvious homology between signal peptides of B. macerans betaglucanase and mesophilic enzymes was observed, with the exception of the motif Leu-Val-Thr conserved within the hydrophobic core sequences. This emphasizes that
Signal peptide and export/n E. coli cells The amino acid sequence from amino acids - 25 to - 1 shows characteristics typical for signal peptides of secretory precursors, i.e. positively charged amino acids at the N-terminus, lengthy hydrophobic core sequence, and an Ala-X-Ala processing site. The N-terminal amino acid sequence of the mature protein purified from E. coli [puC19/34] cells (Fig. 3) has been determined to be GSVFWEPLSY. The beta-glucanase protein isolated from the supernatatant of E. coli [pUC19/34] cells has the same N-terminal sequence. From these findings we conclude that the signal peptide consisting of 25 amino acids is correctly processed in the mature protein (Fig. 2). On the other hand, we were not able to detect any unprocessed beta-glucanase protein in the extracts of re-recombinant E. coli [pUC19/34] cells suggesting that the unprocessed form, if it exists, does not signifi-
ii:;~i!iiiiiiiii!iil;i'iiii~iC
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Fig. 3. Sodium dodecylsulphate/polyacrylamide gel electrophoresis of beta-l,3-1,4-glucanase preparations after each step of the purification process. From left to right: beta-glucanase purified by CMSephadex chromatography (Borriss et al. 1988), 5 pg protein; extract of Escherichia coli (pUC19/34), 42 pg protein; extract of E. coli (pUCI 9/34) after heat treatment (20 min, 60° C)
281 Table 1. Distribution of the beta-glucanase enzyme produced by Escheriehia coli DH5c~[pUC19/34] Cell fraction
% activity (of total activity)
Specific activity (U/mg protein)
23.8 31.2
344 250
24 h b
35.7 32.6
591 697
Cellular 16 h a 24 h b
40.7 36.1
Extracellular 16 h" 24 h b Periplasmic 16 h a
31.2 49.7
Cultures were grown in LB + ampicillin (50 Ixg/ml)at 37° C. Extracellular, periplasmic and cellular fractions were prepared and assayed as described in the Materials and methods. Activities of cytoplasmic glucose-6-phosphate dehydrogenase and periplasmic betalactamase were determined to check possible lysis of cells after 24 h cultivation in LB medium. However, only 7.6% (glucose-6phosphate dehydrogenase) and 2.6% (beta-lactamase) of the total activity were found in the supernatant " Late exponential phase culture b Stationary phase culture even extreme sequence divergence does not impair the function of a signal peptide (Briggs and Gierasch 1986). In order to study the distribution of the beta-glucanase synthesized in E. coli transformants, we isolated extracellular, periplasmic and cellular fractions according to the method described by Cornelis et al. (1982). A significant part of the whole activity has been found in extracellular and periplasmic fractions (Table 1). Furthermore, protein samples prepared from periplasm and extracellular fluid exhibit a very high specific activity (Table 1) and were found to consist of up to 50% of the total beta-glucanase (results not shown). Cytoplasmic glucose-6-phosphate dehydrogenase and periplasmic beta-lactamase activities were also measured as a control for the significance of the data obtained and to exclude cell lysis as a possible reason for the presence of betaglucanase in the extracellular fluid (Table 1). Together with results obtained for the N-terminal sequence of the mature protein, these results suggest that the export mechanism of E. coli seems to accept export signals from Bacillus and correctly processes the protein to the cell periplasm. Similar results have been reported for expression of other extracellular enzyme genes cloned in E. coli (e.g., Cornelis et al. 1982; Borriss et al. 1985). The signal peptide of B. macerans beta-glucanase also seems to direct export of the enzyme in other cells. Beta-glucanase activity has been detected in the supernatant o f yeast cells transformed with the bglM gene, and the N-terminus of the protein purified from the extracellular fluid of this culture has been shown to be identical to that of beta-glucanase secreted by E. coli transformants (Kunze et al. unpublished results). Comparison with other Bacillus beta-glucanases
A high degree of homology has previously been detected between the mesophilic beta-glucanase genes of related
Bacillus species (B. subtilis and B. amyloliquefaciens)
(Hofemeister et al. 1986). About 90% similarity has been found at both the D N A and protein levels. The amino acid sequences of the mature beta-glucanases of B. macerans and B. amyloliquefaciens inferred from the nucleotide sequences are compared in Fig. 4. The processed enzyme from B. macerans is 2 amino acids shorter than the B. amyloliquefaciens and the similar B. subtilis betaglucanases, but striking homologies between those mesophilic enzymes and thermophilic B. macerans beta-glucanase do exist. The extent of similarity of amino acid sequence among the beta-glucanases of B. macerans and B. amyloliquefaciens is 70%, 157 amino acid residues present in both mature beta-glucanases are identical and a further 55 residues can be matched if 4 gaps are introduced in both sequences. Nearby of areas 100% homology were detected in the central and the C-terminal parts of the proteins. In contrast, only relatively weak similarity was found in the N-terminal end of the mature protein of beta-l,3-1,4-glucanases of both Bacillus species (Fig. 4). If amino acid replacement by conservative substitution is taken into account, the overall similarity is increased to nearly 80%. The enhanced thermophilicity of the B. macerans enzyme should be due to its unique amino acid sequences. A slightly higher degree of hydrophobicity is indicated by the hydropathy profile of B. macerans beta-glucanase as compared to the mesophilic enzyme of B. amyloliquefaciens (Fig. 5). The lower content of external, hydrophilic amino acid residues Gln (3/7) and Arg (2/5) might increase the enzyme's internal hydrophobicity so that it folds into a heat-stable form with stronger internal packing in aqueous solution (Imanaka et al. 1986). In addition, molecules with a decreased number of Gln resiAMY MAC
26 26
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Fig. 4. Amino acid sequence homology between mature endo-l,31,4-beta-glucanases from B. macerans and B. amyloliquefaciens. Amino acid residues conserved in both enzymes are boxed. Numbering refers to amino acid sequences of beta-glucanases from B. amyloliquefaciens (amy) and B. macerans (mac)
282
Comparison with T4 lysozyme ~OT ~
=-~l
B. amyloliquefaciens
.
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} Fig. 5. Comparison of the hydrophobicity-hydrophilicity patterns of B. macerans and B. amyloliquefaciens pre-beta-glucanases. The profiles were calculated according to Kyte and Doolittle (1982). The average value for each heptapeptide was plotted against position in the sequence. A r r o w s indicate regions in which the hydrophobicity of B. macerans enzyme is markedly higher than that of the B. amyloliquefaciens enzyme
In contrast to the case of alpha-amylases (Svensson 1988), comparison of different lichenases and endo-beta1,4-glucanases has not revealed any conserved sequences. Limited homologies between cellulase primary structures and the active sites of different lysozymes have, however, been noted (Teeri et al. 1987). There is some evidence that endo-glucanases act like lysozymes, which split glycosidic bonds by an acid catalysis mechanism (Vernon and Banks 1963) involving carboxyl groups at the active site (Legler and Bause 1973; Clarke and Yaguchi 1985). Inhibitor studies recently performed with lichenan-hydrolysing glucanases from B. subtilis, Streptomyces and Rhizopus arrhizus support the hypothesis that, despite the differences that exist in active site geometry between the various endo-glucanases, aspartate and/or glutamate are involved in binding of substrate and catalysis of the hydrolysis reaction (Hoj et al. 1989). Sequences surrounding the catalytic residues Glu (11) and Asp (20) of T4 lysozyme (Weaver and Matthews 1987) were aligned with the whole amino acid sequence of B. macerans beta-glucanase using the dot matrix approach. Only a short sequence between amino acids 126 and 161 was found to be related. Interestingly, this sequence is highly conserved in all Bacillus /%glucanase enzymes and contains the putative active residues Glu (128) and Asp (137) within the motif DE - X7 - KDT - X2/6 - YYT
dues might be less affected by irreversible thermoinactivation caused by deamidation reactions. Comparison with functionally related enzymes
It has recently been shown that Bacillus cellulase (endobeta-l,4-glucanase) genes are derived from a common ancestral gene (Fukumori et al. 1989)• Moreover, comparison of the amino acid sequence of Clostridium acetobutylicum endo-glucanase with the sequences of the two 1,4-beta-glucanases from alkalophilic Bacillus strain N-4 (Fukumori et al. 1986) and with those of the genes coding for the 1,4-beta-glucanases from B. subtilis strains P A P l l 5 (Mackey et al. 1986) and D L G (Robson and Chambliss 1987) (which exhibit 93.4% homology) showed in all cases about 45% homology (Zappe et al. 1988). This indicates extensive homology of the nucleotide and amino acid sequences of endo-l,4-beta-glucanase genes from Clostridium spp. and Bacillus spp• Comparison of the amino acid sequence of bglM with those of the bacterial beta-l,4-glucanases mentioned above revealed, however, no discernible homology• Moreover, plant endo-beta-l,3-1,4 glucanases do not share any conserved sequences with the corresponding Bacillus glucanases (Fincher et al. 1986). Thus, we conclude that Bacillus endo-beta-l,3-1,4-glucanases evolved independently of their plant counterparts and of other microbial endo-beta-glucanases.
(where X is any amino acid and subscript 2/6 indicates 2 or 6 residues). This would position the active site of B. macerans beta-glucanase within the central region of the enzyme. Figure 6 shows a compilation of the limited amino acid homologies found between phage T4 lysozyme and this conserved area in Bacillus beta-glucanases. Other examples of limited sequence homology between glucanases and the active sites of lysozymes have been reported for beta-glucanases of Schizophyllum commune (Clarke and Yaguchi 1985), Trichoderma reesei (Teeri et al. 1987), and several fungal and bacterial betaglucosidases (Graebnitz et al. 1989). Our findings support the notion that Bacillus endo-beta-l,3-1,4-glucanases might act, like lysozyme, by an acid catalysis mecha-
11
20
24
31
36
Fig. 6. Alignment of the beta-glucanase sequence of B. rnacerans with the active site sequence of phage T4 lysozyme (T4-L). The T4-L sequence shows the region of established active site residues Glu-ll and Asp-20 (Weaver and Matthews 1987). Bgl shows the region in the B. maeerans beta-glucanase amino acid sequence (residues 126-160 of the pre-beta-glucanase) which shows limited homology to active site regions of T4-L. Identical positions are b o x e d and conservative exchanges are connected by broken lines. Asterisks indicate the active site residues, Asp and Glu in T4-L
283 n i s m as d o o t h e r e n d o - a n d exo-glucosidases. H o w e v e r , w h e t h e r o r n o t t h o s e r e g i o n s w i t h l i m i t e d h o m o l o g y to T4 l y s o z y m e c o r r e s p o n d to a n active site s h o u l d be tested b y s i t e - d i r e c t e d m u t a g e n e s i s a n d active centre m o d i f i c a t i o m s t u d i e s as p r o p o s e d b y Teeri et al. (1987). Acknowledgements. Part of this work has been performed within
the framwork of cooperation between the Academies of Sciences of Finland and of the German Democratic Republic. R.B. would like especially to thank Peter Meyer for introduction techniques of DNA sequencing and all members of the Recombinant DNA Group in the Department of Biochemistry, University of Turku, for support and hospitality. We wish to acknowledge the contribution made by Dr. Gotthard Kunze (site-directed mutagenesis) and Dr. Karl Kristian Thomsen, Carlsberg Laboratory (determination of N-terminal amino acids). The technical assistance of Mrs. I. Kaeppler is thankfully acknowledged. Drs. R. Rieger and R. Mendel are thanked for critical reading of the manuscript.
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