Identification and Characterization of Bacterial Cutinase

Cutinase, which exists in both fungi and bacteria, catalyzes the cleavage of the ester bonds of cutin. Fungal cutinases have been extensively studied, however, reports on bacterial cutinases have been limited due to the lack of knowledge concerning the identity of their open reading frames. In the present study, the cutinase from Thermobifida fusca was induced by cutin and purified to homogeneity by following p-nitrophenyl butyrate hydrolyzing activity. Peptide mass fingerprinting analysis of the wild-type enzyme matched two proteins, Tfu₀883 and Tfu₀882, which are 93% identical in sequence. Both proteins were cloned and overexpressed in their mature form. Recombinant Tfu₀883 and Tfu₀882 display very similar enzymatic properties and were confirmed to be cutinases by their capability to hydrolyze the ester bonds of cutin. Comparative characterization of Fusarium solani pisi and T. fusca cutinases indicated that they have similar substrate specificity and catalytic properties except that the T. fusca enzymes are thermally more stable. Homology modeling revealed that T. fusca cutinases adopt an α/β-hydrolase fold that exhibits both similarities and variations from the fungal cutinase structure. A serine hydrolase catalytic mechanism involving a Ser170-His248-Asp216 (Tfu₀883 numbering) catalytic triad was supported by active site-directed inhibition studies and mutational analyses. This is the first report of cutinase encoding genes from bacterial sources. Cutinase, which exists in both fungi and bacteria, catalyzes the cleavage of the ester bonds of cutin. Fungal cutinases have been extensively studied, however, reports on bacterial cutinases have been limited due to the lack of knowledge concerning the identity of their open reading frames. In the present study, the cutinase from Thermobifida fusca was induced by cutin and purified to homogeneity by following p-nitrophenyl butyrate hydrolyzing activity. Peptide mass fingerprinting analysis of the wild-type enzyme matched two proteins, Tfu₀883 and Tfu₀882, which are 93% identical in sequence. Both proteins were cloned and overexpressed in their mature form. Recombinant Tfu₀883 and Tfu₀882 display very similar enzymatic properties and were confirmed to be cutinases by their capability to hydrolyze the ester bonds of cutin. Comparative characterization of Fusarium solani pisi and T. fusca cutinases indicated that they have similar substrate specificity and catalytic properties except that the T. fusca enzymes are thermally more stable. Homology modeling revealed that T. fusca cutinases adopt an α/β-hydrolase fold that exhibits both similarities and variations from the fungal cutinase structure. A serine hydrolase catalytic mechanism involving a Ser170-His248-Asp216 (Tfu₀883 numbering) catalytic triad was supported by active site-directed inhibition studies and mutational analyses. This is the first report of cutinase encoding genes from bacterial sources. Cutin is a major component of the plant cuticle. It is an insoluble lipid-polyester formed primarily from C16 and C18 hydroxy and epoxy fatty acids (Fig. 1) (1Fett W. F. Gerard H. C. Moreau R. A. Osman S. F. Jones L. E. Appl. Environ. Microb. 1992; 58: 2123-2130Crossref PubMed Google Scholar, 2Walton T. J. Kolattukudy P. E. Biochemistry. 1972; 11: 1885-1896Crossref PubMed Scopus (164) Google Scholar, 3Purdy R. E. Kolattukudy P. E. Arch. Biochem. Biophys. 1973; 159: 61-69Crossref PubMed Scopus (79) Google Scholar). Although the precise composition of cutin varies among species, all cutins contain hydroxy fatty acids as the base units (4Gerard H. C. Osman S. F. Fett W. F. Moreau R. A. Phytochem. Analysis. 1992; 3: 139-144Crossref Scopus (23) Google Scholar). Cutinases are inducible extracellular enzymes secreted by microorganisms that are capable of degrading plant cell walls. They catalyze the cleavage of ester bonds of cutin, resulting in the release of cutin monomers (5Ferreira B. S. Calado C. R. van Keulen F. Fonseca L. P. Cabral J. M. da Fonseca M. M. Appl. Microbiol. Biotechnol. 2003; 61: 69-76Crossref PubMed Scopus (26) Google Scholar, 6Sebastian J. Kolattukudy P. E. Arch. Biochem. Biophys. 1988; 263: 77-85Crossref PubMed Scopus (34) Google Scholar, 7Wang G. Y. Michailides T. J. Hammock B. D. Lee Y. M. Bostock R. M. Fungal Genet. Biol. 2002; 35: 261-276Crossref PubMed Scopus (48) Google Scholar, 8Carvalho C. M. Aires-Barros M. R. Cabral J. M. Biotechnol. Bioeng. 1999; 66: 17-34Crossref PubMed Scopus (198) Google Scholar). Cutinases have been found in both fungi and bacteria; however, studies have focused on fungal cutinases, including Fusarium solani pisi (9Egmond M. R. de Vlieg J. Biochimie (Paris). 2000; 82: 1015-1021Crossref PubMed Scopus (117) Google Scholar), Monilinia fructicola (7Wang G. Y. Michailides T. J. Hammock B. D. Lee Y. M. Bostock R. M. Fungal Genet. Biol. 2002; 35: 261-276Crossref PubMed Scopus (48) Google Scholar, 10Wang G. Y. Michailides T. J. Hammock B. D. Lee Y. M. Bostock R. M. Arch. Biochem. Biophys. 2000; 382: 31-38Crossref PubMed Scopus (34) Google Scholar), Botrytis cinerea (11Gindro K. Pezet R. FEMS Microbiol. Lett. 1999; 171: 239-243Crossref PubMed Google Scholar), and Aspergillus oryzae (12Maeda H. Yamagata Y. Abe K. Hasegawa F. Machida M. Ishioka R. Gomi K. Nakajima T. Appl. Microbiol. Biotechnol. 2005; 67: 778-788Crossref PubMed Scopus (181) Google Scholar). Cutinase from F. solani pisi, for example, has been extensively investigated by biochemical and structural approaches (13Cunha M. T. Costa M. J. Calado C. R. Fonseca L. P. Aires-Barros M. R. Cabral J. M. J. Biotechnol. 2003; 100: 55-64Crossref PubMed Scopus (33) Google Scholar, 14Longhi S. Cambillau C. Biochim. Biophys. Acta. 1999; 1441: 185-196Crossref PubMed Scopus (110) Google Scholar). X-ray crystallography studies of F. solani pisi cutinase revealed that it belongs to the α/β-hydrolase fold superfamily, and contains the characteristic GXSXG motif as well as a Ser-His-Asp catalytic triad (9Egmond M. R. de Vlieg J. Biochimie (Paris). 2000; 82: 1015-1021Crossref PubMed Scopus (117) Google Scholar). Interestingly, fungal cutinases hydrolyze not only cutin, but also insoluble triglycerides and soluble esters such as p-nitrophenyl butyrate (pNPB). 3The abbreviations used are: pNPBp-nitrophenyl butyrateSeLStreptomyces exfoliates lipasePMSFphenylmethylsulfonyl fluoridePsLPseudomonas sp. MIS38 lipaseGC/MSgas chromatography/mass spectrometry. Thus, fungal cutinases are considered as being intermediate between lipases and esterases (8Carvalho C. M. Aires-Barros M. R. Cabral J. M. Biotechnol. Bioeng. 1999; 66: 17-34Crossref PubMed Scopus (198) Google Scholar, 9Egmond M. R. de Vlieg J. Biochimie (Paris). 2000; 82: 1015-1021Crossref PubMed Scopus (117) Google Scholar, 14Longhi S. Cambillau C. Biochim. Biophys. Acta. 1999; 1441: 185-196Crossref PubMed Scopus (110) Google Scholar). Recently, it was found that F. solani pisi cutinase can hydrolyze and thus improve the surface properties of synthetic fibers such as polyethyleneterephthalate fibers in an environmentally friendly way (15Araujo R. Silva C. O'Neill A. Micaelo N. Guebitz G. Soare C. M. Casal M. Cavaco-Paulo A. J. Biotechnol. 2007; 128: 849-857Crossref PubMed Scopus (144) Google Scholar). Such versatile hydrolytic activities of cutinases suggest that they may have promising applications in chemical, textile, and other industries (16Alisch-Mark M. Herrmann A. Zimmermann W. Biotechnol. Lett. 2006; 28: 681-685Crossref PubMed Scopus (101) Google Scholar, 17Vertommen M. A. Nierstrasz V. A. Veer M. Warmoeskerken M. M. J. Biotechnol. 2005; 120: 376-386Crossref PubMed Scopus (240) Google Scholar, 18Degani O. Gepstein S. Dosoretz C. G. Appl. Biochem. Biotechnol. 2002; 102–103: 277-289Crossref PubMed Scopus (95) Google Scholar). p-nitrophenyl butyrate Streptomyces exfoliates lipase phenylmethylsulfonyl fluoride Pseudomonas sp. MIS38 lipase gas chromatography/mass spectrometry. In contrast to fungal cutinases, little is known about cutinases from bacterial sources. There are a few reports of bacterial cutinases from Streptomyces scabies (19Lin T. S. Kolattukudy P. E. Plant Pathol. 1980; 17: 1-15Google Scholar), Thermobifida fusca (20Fett W. F. Wijey C. Moreau R. A. Osman S. F. J. Appl. Microbiol. 1999; 86: 561-568Crossref Scopus (53) Google Scholar), and Pseudomonas putida (6Sebastian J. Kolattukudy P. E. Arch. Biochem. Biophys. 1988; 263: 77-85Crossref PubMed Scopus (34) Google Scholar, 21Sebastian J. Chandra A. K. Kolattukudy P. E. J. Bacteriol. 1987; 169: 131-136Crossref PubMed Google Scholar) ; however, these studies were limited to screening of enzyme-producing strains and initial characterization on crude enzyme preparations. Most critically, no cutinase open reading frame has been identified in bacteria, thus it is impossible to obtain sufficient amounts of pure recombinant enzymes for in depth studies. In the present study, for the first time, the gene responsible for the expression of bacterial cutinase was identified. Two cutin-induced wild-type pNPB hydrolases were isolated from T. fusca. Peptide mass fingerprinting and data base search of these hydrolases revealed that they are encoded by open reading frames, Tfu₀882 and Tfu₀883, respectively. The two enzymes are 93% identical in amino acid sequence. The mature forms of both enzymes were cloned, expressed, and purified to homogeneity, and were confirmed to function as cutinase. The biochemical properties of T. fusca cutinases were investigated and compared with their F. solani pisi counterpart. Molecular modeling, inhibition studies, and mutational analysis shed light on the mechanism and key residues in catalysis. Materials—A Bacillus subtilis strain harboring the plasmid pBSMuL3 containing the F. solani pisi cutinase was kindly provided by Dr. Thorsten Eggert. The EZ-10 Spin Column Plasmid Mini-Preps kit, agarose gel DNA purification kit, restriction enzymes, and T4 DNA ligase were obtained from TakaRa Biotechnology Co. Ltd. The plasmids pMD18T-simple and pET20b (+) were obtained from Novagen. Sodium taurodeoxycholate, phenylmethylsulfonyl fluoride (PMSF), bis- (trimethylsilyl) trifluoroacetamide, pNPB, and Pseudomonas sp. MIS38 lipase (PsL) were obtained from Sigma. Other chemicals were obtained from Sinopharm Chemical Reagent Co. Ltd. Phenyl-Sepharose FF and DEAE-Sepharose FF resins were obtained from Amersham Biosciences. Cellulase and pectinase were obtained from Wuxi Boli Biotechnologies Co. Ltd. DNA primers were synthesized by Shanghai Sangon Biological Engineering Technology Scopus Google Scholar). of enzyme is as the of 1 of The was in a of 1 containing pNPB the and the 10 NaCl, and 50 sodium taurodeoxycholate, 8. 0) at 20 The was by the of The of pNPB was for the of the at Cutinase was as with the following (20Fett W. F. Wijey C. Moreau R. A. Osman S. F. J. Appl. Microbiol. 1999; 86: 561-568Crossref Scopus (53) Google Scholar). cutin, from mature as T. J. Kolattukudy P. E. Biochemistry. 1972; 11: 1885-1896Crossref PubMed Scopus (164) Google Scholar), as the a a of enzyme and of cutin were a (pH 8. 0) in a of 10 The was for in a at the the of the cutin was by The resulting was with acid and the cutin monomers were with The soluble was and a of The cutin monomers were to their esters and with S. F. Fett W. F. N. J. 1999; PubMed Scopus Google Scholar). The esters were in and by on a with as °C for 5 to and °C for was as S. C. van M. O. FEMS Microbiol. PubMed Google with the following as the The contained of (pH and 2 of The was by the enzyme to the and by 7. 5 of The fatty acids were by of lipase was as the release of 1 of fatty acid and of of enzyme was between 20 and the was performed in a containing 20 10 NaCl, and 50 sodium at pNPB as the the of is the were to at the The enzyme was by the at a for 2 to it to the of lipase the was performed in (pH 8. 0) as the The of enzyme was between and (pH 20 (pH of of the enzyme was by the enzyme in 20 (pH 8. 0) at of enzymes were and for pNPB as the of between Tfu₀882 and the of a between the two T. fusca cutinases in cutin Tfu₀882 and Tfu₀883 were as a with cutin in (pH 8. 0) at were and for fatty acids by with The fatty acid monomers of were identified by as of of the enzyme was performed by the enzyme with 1 in 20 (pH at °C for amounts of The were by as The enzyme was by pNPB as the was not to the these in not The of inhibition was performed an of 10 of Cutinase were by the The of Tfu₀882 was as of Tfu₀883 was as were by DNA The were and purified the as for the recombinant T. fusca The of the purified was pNPB as was performed the from was performed on a The gel was with was by the Purification of a Cutin-induced pNPB Hydrolase from T. major to the of wild-type bacterial cutinase has been the in cutin to enzyme pNPB as the substrate on the following cutinase is inducible by cutin (1Fett W. F. Gerard H. C. Moreau R. A. Osman S. F. Jones L. E. Appl. Environ. Microb. 1992; 58: 2123-2130Crossref PubMed Google Scholar), is to the and can hydrolyze pNPB S. Cambillau C. Biochim. Biophys. Acta. 1999; 1441: 185-196Crossref PubMed Scopus (110) Google Scholar). found that pNPB hydrolase in T. fusca about in the of cutin, no pNPB hydrolase was in the of cutin. analysis of proteins in the indicated that a of was the major induced by cutin that it be a for cutinase by the of pNPB, a pNPB hydrolase was purified from cutin-induced of T. fusca and The purified enzyme a of for pNPB analysis that pNPB hydrolase to the induced by cutin Cutin-induced pNPB Hydrolase T. fusca pNPB hydrolase also cutinase the was for of cutin. recombinant cutinase from F. solani pisi was purified and the in both enzymes for cutin fatty acid example, the of C16 and C18 fatty acids monomers enzymatic were for T. fusca pNPB hydrolase and for F. solani pisi cutinase. the of hydroxy fatty acid is for T. fusca pNPB hydrolase and for F. solani pisi cutinase indicated that the cutin-induced pNPB hydrolase from T. fusca as a cutinase. of T. fusca Cutinase the gene encoding T. fusca the to cutin-induced pNPB hydrolase was from the gel and to The resulting were by mass spectrometry. The mass data were used to the data base resulting in two Tfu₀883 and Tfu₀882 the matched were by both proteins, were for Tfu₀883, and 1 was for Tfu₀882 amino acid sequencing of the only a of which to residues of Tfu₀882 and residues of that residues of Tfu₀882 and residues of Tfu₀883 are their both Tfu₀882 and Tfu₀883 are amino acids in and a identity of It is to be that both proteins were as lipase in the data base and contain the a characteristic of the α/β-hydrolase fold M. Biol. 1999; PubMed Scopus Google of Tfu₀882 and for for for mature in a of mass data Tfu₀883 and for for by Tfu₀882 and in a and Purification of Tfu₀882 and genes encoding the mature forms of Tfu₀882 and Tfu₀883 were for The for contains a and which the proteins to be The pNPB hydrolyzing in the of cells harboring plasmid was which was that of cells and that of cutin-induced T. fusca The pNPB hydrolyzing in the of cells harboring plasmid was which was that of cells and that of cutin-induced T. fusca The recombinant enzymes were purified to homogeneity in a by and a of for Tfu₀883 and for of Recombinant Tfu₀882 and the fungal cutinase from F. solani pisi can hydrolyze both insoluble triglycerides and soluble esters R. E. Kolattukudy P. E. Arch. Biochem. Biophys. 1973; 159: 61-69Crossref PubMed Scopus (79) Google Scholar), Tfu₀882 and Tfu₀883 were first for the of and pNPB, and compared with their fungal counterpart. in all enzymes and Tfu₀882 and Tfu₀883 an at °C for both and pNPB, F. solani pisi cutinase an at °C for and °C for pNPB (Fig. all enzymes a of about for the two (Fig. of cutinase. and Tfu₀882 pNPB and as and Tfu₀883 pNPB and as and F. solani pisi cutinase pNPB and as respectively. was at °C for F. solani pisi cutinase and °C for Tfu₀882 and Tfu₀883, in (pH 20 (pH to the of The of Tfu₀882, Tfu₀883, and F. solani pisi cutinase were at both and °C (Fig. Interestingly, the of Tfu₀882 and Tfu₀883 at both F. solani pisi cutinase a similar initial at but a at T. fusca cutinases with activities of at °C at In F. solani pisi cutinase was with of at °C 5 at to Tfu₀882 and Tfu₀883 can hydrolyze cutin, both enzymes and the fungal cutinase were their and in the C16 and C18 fatty acid monomers enzymatic were for Tfu₀883, for Tfu₀882, and for F. solani pisi cutinase. The hydroxy fatty acids that are in cutin were for Tfu₀883, for Tfu₀882, and for F. solani pisi cutinase. confirmed that both Tfu₀882 and Tfu₀883 are cutin from cutin by recombinant cutinases and a bacterial solani cutinase fusca cutinase fusca cutinase sp. MIS38 lipase not not in a A as to is a between the two T. fusca enzymes in the of cutin. Tfu₀882, Tfu₀883, a containing the amounts of both enzymes were with cutin the the of fatty acids by the enzyme was identical to the of from enzymes (Fig. the the In analysis of the revealed no in the and of fatty acid monomers by Tfu₀882, Tfu₀883, the of not Thus, it is no between Tfu₀882 and Tfu₀883 in cutin Homology of T. fusca Tfu₀882 and Tfu₀883 are similar in only Tfu₀883, was for and A search of the data base with Tfu₀883 revealed that are as lipase The was found with Streptomyces exfoliates lipase has been at Y. C. W. H. J. K. PubMed Scopus Google Scholar). the mechanism of bacterial a of Tfu₀883 on was by the modeling T. J. N. 2003; PubMed Scopus Google Scholar). The exhibits an α/β-hydrolase fold of a on both by (Fig. In similar to Tfu₀883 contains an This also revealed a Ser170-His248-Asp216 catalytic triad in which the is in a the which is in the M. Biol. 1999; PubMed Scopus Google Scholar). The is to be formed by the of and similar to fungal the enzyme not contain a that is in lipases S. C. van M. O. FEMS Microbiol. PubMed Google Scholar), resulting in a serine that is to the (Fig. from other bacterial to not secreted not in a and of T. fusca the enzyme a catalytic triad the of a serine hydrolase on enzyme and investigated the of the serine on catalysis. in the activities of the two cutinases were by of for Tfu₀882 and of for Tfu₀883, with a of for Tfu₀882 and for Tfu₀883, of the catalytic serine for both the enzymes were with for 10 inhibition of Tfu₀882 and Tfu₀883 were at about and respectively. of the catalytic serine in Tfu₀882 and in to their Although fungal cutinase has been extensively for the identity of bacterial cutinase has a In the present study, of that Tfu₀882 and Tfu₀883 from the T. fusca function as a was secreted the T. fusca cutin and cutinase mass fingerprinting analysis and amino acid sequencing of the matched two proteins, Tfu₀882 and Tfu₀883, which are 93% identical in both Tfu₀882 and Tfu₀883 are to hydrolyze cutin, resulting in of fungal cutinase. their it is not that Tfu₀882 and Tfu₀883 have similar properties and were thus purification of the wild-type both Tfu₀882 and Tfu₀883 are versatile in that they can both insoluble triglycerides and soluble esters as substrate in to cutin. the two enzymes similar and and suggest that they may be as cutinase in T. fusca. Although Tfu₀882 and Tfu₀883 are in the due to a gene they not to be in an as by the 2005; PubMed Scopus Google Scholar). is no between the two enzymes in cutin (Fig. studies are to are two genes for T. fusca cutinase. analysis that T. fusca cutinases to the α/β-hydrolase fold M. Biol. 1999; PubMed Scopus Google Scholar). in a of hydrolytic activities M. 2000; PubMed Scopus Google Scholar), however, they all adopt a fold and are to have from a M. Biol. 1999; PubMed Scopus Google Scholar). the of Tfu₀883 a α/β-hydrolase fold with a Ser170-His248-Asp216 triad and a (Fig. a serine hydrolase mechanism involving two and an intermediate G. A. Biochem. PubMed Scopus Google Scholar). mechanism was supported by inhibition of Tfu₀882 and Tfu₀883 (Fig. as well as site-directed of the catalytic serine in both A of the T. fusca cutinase is that the enzyme not contain a in lipases C. H. K. Y. K. S. Lett. 2007; PubMed Scopus Google Scholar, H. A. S. Biochemistry. 2000; PubMed Scopus Google Scholar, F. PubMed Scopus Google Scholar, F. J. Biol. PubMed Scopus Google Scholar), and serine to the (Fig. In the is to be in the in which it in to at the S. C. van M. O. FEMS Microbiol. PubMed Google Scholar). The of such a that the T. fusca cutinases to a of from the the other the open active of T. fusca cutinase the of the enzyme to cutin. studies, crystallography studies of cutinase may the structural of substrate Comparative biochemical characterization of bacterial and fungal cutinases indicated that they have similar substrate specificity and catalytic properties except that T. fusca cutinases This may T. fusca cutinases more for Although both T. fusca and F. solani pisi cutinases to the and contain an open active the bacterial enzymes have and no to the fungal the fungal cutinase contains the two of the α/β-hydrolase fold the of Thus, the bacterial and fungal enzymes have and are to be and cutinase respectively. Tfu₀883 was to be a hydrolase capable of degrading by the K. J. 2005; PubMed Scopus Google Scholar). In their studies, an extracellular hydrolase responsible for the of was purified from the of T. fusca and for amino acid sequence. The of their enzyme is identical to that of Although was for they that of enzyme as a cutinase is a lipase Pseudomonas sp. MIS38 lipase (PsL) was also to hydrolyze their cutin and release fatty acids by a to cutinase by to the fatty acid It that enzyme no cutinase C16 and C18 fatty the characteristic cutin were in the enzymatic The fatty acids found in the enzymatic be the of that in the cutin In have found that it is to obtain cutin that is of which also be the in the by and K. J. 2005; PubMed Scopus Google Scholar). the catalytic residues of are the which is from the active in and the fungal cutinase (Fig. these indicated that from in and be as a cutinase. In the present has that the Tfu₀882 and Tfu₀883 from the T. fusca are Although their catalytic properties and are similar to the fungal cutinases, and structural the T. fusca enzymes a of cutinases from the fungal their applications in the of the bacterial cutinase encoding genes an that to other of the for with