Characterization of a Novel Ser-cisSer-Lys Catalytic Triad in Comparison with the Classical Ser-His-Asp Triad

Abstract

Amidase signature family enzymes, which are widespread in nature, contain a newly identified Ser-cisSer-Lys catalytic triad in which the peptide bond between Ser131 and the preceding residue Gly130 is in a cis configuration. In order to characterize the property of the novel triad, we have determined the structures of five mutant malonamidase E2 enzymes that contain a Cys-cisSer-Lys, Ser-cisAla-Lys, or Ser-cisSer-Ala triad or a substitution of Gly130 with alanine. Cysteine cannot replace the role of Ser155 due to a hyper-reactivity of the residue, which results in the modification of the cysteine to cysteinyl sulfinic acid, most likely inside the expression host cells. The lysine residue plays a structural as well as a catalytic role, since the substitution of the residue with alanine disrupts the active site structure completely. The two observations are in sharp contrast with the consequences of the corresponding substitutions in the classical Ser-His-Asp triad. Structural data on the mutant containing the Ser-cisAla-Lys triad convincingly suggest that Ser131 plays an analogous catalytic role as the histidine of the Ser-His-Asp triad. The unusual cis configuration of Ser131 appears essential for the precise contacts of this residue with the other triad residues, as indicated by the near invariance of the preceding glycine residue (Gly130), structural data on the G130A mutant, and by a modeling experiment. The data provide a deep understanding of the role of each residue of the new triad at the atomic level and demonstrate that the new triad is a catalytic device distinctively different from the classical triad or its variants. Amidase signature family enzymes, which are widespread in nature, contain a newly identified Ser-cisSer-Lys catalytic triad in which the peptide bond between Ser131 and the preceding residue Gly130 is in a cis configuration. In order to characterize the property of the novel triad, we have determined the structures of five mutant malonamidase E2 enzymes that contain a Cys-cisSer-Lys, Ser-cisAla-Lys, or Ser-cisSer-Ala triad or a substitution of Gly130 with alanine. Cysteine cannot replace the role of Ser155 due to a hyper-reactivity of the residue, which results in the modification of the cysteine to cysteinyl sulfinic acid, most likely inside the expression host cells. The lysine residue plays a structural as well as a catalytic role, since the substitution of the residue with alanine disrupts the active site structure completely. The two observations are in sharp contrast with the consequences of the corresponding substitutions in the classical Ser-His-Asp triad. Structural data on the mutant containing the Ser-cisAla-Lys triad convincingly suggest that Ser131 plays an analogous catalytic role as the histidine of the Ser-His-Asp triad. The unusual cis configuration of Ser131 appears essential for the precise contacts of this residue with the other triad residues, as indicated by the near invariance of the preceding glycine residue (Gly130), structural data on the G130A mutant, and by a modeling experiment. The data provide a deep understanding of the role of each residue of the new triad at the atomic level and demonstrate that the new triad is a catalytic device distinctively different from the classical triad or its variants. A large group of enzymes, designated as amidase signature (AS) 1The abbreviations used are: AS, amidase signature; MAE2, malonamidase E2; Rmsd, root mean-squared deviation. 1The abbreviations used are: AS, amidase signature; MAE2, malonamidase E2; Rmsd, root mean-squared deviation. family enzymes, are widely distributed throughout the hierarchy of living organisms. A search against the Swiss-Prot data base with the BLAST algorithm (1Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Google Scholar) identifies over 200 different AS family proteins from 90 different organisms. These enzymes are characterized by the AS sequence, which is a conserved stretch of ∼130 amino acids (2Mayaux J.F. Cerbelaud E. Soubrier F. Yeh P. Blanche F. Petre D. J. Bacteriol. 1991; 173: 6694-6704Google Scholar, 3Chebrou H. Bigey F. Arnaud A. Galzy P. Biochim. Biophys. Acta. 1996; 5: 285-293Google Scholar). The identified biochemical function of the AS family enzymes has commonly been the hydrolysis of the amide bond (CO–NH2). The known biological functions of the AS family enzymes vary widely, including the formation of Gln-tRNAGln by the transamidation of misacylated Glu-tRNAGln via amidolysis of glutamine in many different bacteria (4Schon A. Kannangara C.G. Gough S. Soll D. Nature. 1988; 331: 187-190Google Scholar, 5Curnow A.W. Hong K. Yuan R. Kim S. Martins O. Winkler W. Henkin T.M. Soll D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11819-11826Google Scholar), catabolism of neuromodulatory fatty acid amides in mammals (6Cravatt B.F. Giang D.K. Mayfield S.P. Boger D.L. Lerner R.A. Gilula N.B. Nature. 1996; 384: 83-87Google Scholar, 7Omeir R.L. Arreaza G. Deutsch D.G. Biochem. Biophys. Res. Commun. 1999; 264: 316-320Google Scholar, 8Koutek B. Prestwich G.D. Howlett A.C. Chin S.A. Salehani D. Akhavan N. Deutsch D.G. J. Biol. Chem. 1994; 269: 22937-22940Google Scholar, 9Deutsch D.G. Ueda N. Yamamoto S. Prostaglandins Leukot. Ess. Fatty Acids. 2002; 66: 201-210Google Scholar), the formation of indole-3-acetic acid in pathogenic plant bacteria (10Gaffney T.D. da Costa e Silva O. Yamada T. Kosuge T. J. Bacteriol. 1990; 172: 5593-5601Google Scholar), and the metabolic turnover of carbon/nitrogen-containing compounds in both prokaryotes and eukaryotes (2Mayaux J.F. Cerbelaud E. Soubrier F. Yeh P. Blanche F. Petre D. J. Bacteriol. 1991; 173: 6694-6704Google Scholar, 11Gomi K. Kitamoto K. Kumagai C. Gene (Amst.). 1991; 108: 91-98Google Scholar). One member of this family, nitrile hydratase, is used in the industrial scale production of acrylamide and nicotinamide (12Yamada H. Kobayashi M. Biosci. Biotechnol. Biochem. 1996; 60: 1391-1400Google Scholar). Despite the functional variety and the wide evolutionary distribution of the AS family enzymes, which rival that of the other classical serine hydrolases, e.g. trypsin-like serine protease family containing about 270 different members in the Swiss-Prot data base, little is known about the structure-function relationship of the AS family enzymes. Recently, we reported the structure of malonamidase E2 (MAE2) from Brady-rhizobium japonicum, the first structure of an AS family enzyme (13Shin S. Lee T.H. Ha N.C. Koo H.M. Kim S.Y. Lee H.S. Kim Y.S. Oh B.H. EMBO J. 2002; 21: 2509-2516Google Scholar). The structure revealed a novel catalytic triad Ser-cisSer-Lys. The peptide bond between the second serine, Ser131, and the preceding residue, Gly130, is in an unusual cis configuration. The novel catalytic triad had not been observed in any other known hydrolytic enzymes. Subsequently, the structures of two other AS family enzymes have been reported: peptide amidase of Stenotrophomonas maltophilia (14Labahn J. Neumann S. Buldt G. Kula M. Granzin J. J. Mol. Biol. 2002; 322: 1053-1064Google Scholar) and rat fatty acid amide hydrolase (15Bracey M.H. Hanson M.A. Masuda K.R. Stevens R.C. Cravatt B.F. Science. 2002; 298: 1793-1796Google Scholar), both of which revealed the cis configuration of Ser131. The triad residues, found exclusively on the AS sequences, are absolutely conserved, and no variation of the chemical makeup of the triad has been observed. This is in a sharp contrast with the classical Ser-His-Asp triad, which is often found in several completely dissimilar protein folds (16Dodson G. Wlodawer A. Trends Biochem. Sci. 1998; 23: 347-352Google Scholar), and whose variations are found in similar protein folds, such as the Cys-His-Glu triad in human rhinovirus 3C protease, an α-chymotrypsin-like protease (17Matthews D.A. Smith W.W. Ferre R.A. Condon B. Budahazi G. Sisson W. Villafranca J.E. Janson C.A. McElroy H.E. Gribskov C.L. Worland S. Cell. 1994; 77: 761-771Google Scholar) and Ser-His-Glu triad in a fungal lipase (18Schrag J.D. Li Y.G. Wu S. Cygler M. Nature. 1991; 351: 761-764Google Scholar). While the classical triad has been characterized in minute detail, little is known about the characteristics of the Ser-cisSer-Lys triad. In this report, we provide a structural basis for understanding the invariability of the chemical makeup of the novel triad and delineate the role of each constituent amino acid in detail. This study led to the conclusions that the novel triad is a catalytic device distinctively different from the classical triad or its variants, and for this reason, that the AS family is evolutionary distinct from any other known hydrolase families. Site-directed Mutagenesis and Protein Purification—S155C, S131A, K62A, G130A, and T150A MAE2 mutants were created using the QuikChange kit (Stratagene). The plasmids containing each of the mutant MAE2 genes was transformed into Escherichia coli strain BL21(DE3). The expression of each of the MAE2 mutants was induced by 1 mm isopropyl-β-d-thiogalactopyranoside at an optical density of 0.6–0.8 at 37 °C for 16 h except for the G130A mutant, which required the induction at 24 °C. Bacterial lysates were prepared by sonication in buffer A, 20 mm Tris-HCl solution (pH 7.4) containing 15% glycerol. After centrifugation, the supernatant was applied to a HiTrap™ Blue HP column (Amersham Biosciences) and eluted with a linear gradient from 0 to 2 m of NaCl in buffer A. The fractions containing MAE2 were dialyzed against buffer B, 20 mm Tris-HCl solution (pH 7.4) containing 2 m NaCl. The dialyzed protein sample was loaded onto the RESOURCE™ PHE column (Amersham Biosciences) pre-equilibrated with buffer B containing 2 mm ZnSO4. The unbound fraction containing MAE2 was concentrated and subsequently loaded on a Superdex 200 HR 20/60 column (Amersham Biosciences) pre-equilibrated with 20 mm Tris-HCl (pH 7.4). The peak fractions were concentrated to 10 mg/ml using centriprep-10K (Amicon). For the purification of the S155C mutant, we included 1 mm dithiothreitol in all the buffers used. Crystallization, X-ray Data Collection, and Structure Determination—The crystals of the MAE2 mutants were obtained in droplets containing 1.5 μl of protein sample (10 mg/ml) and an equal volume of precipitant solution containing 20% polyethylene glycol 1000 and 0.1 m Tris-HCl (pH 7.0). The crystals of the S131A MAE2 mutant in complex with malonamate or with malonate were obtained by the cocrystallization method in the presence of 5 mm malonamate or 15 mm malonate using the same precipitant solution. All crystals were flash-cooled in the stream of cooled nitrogen gas (100 K) prior to x-ray data collection. All data sets were collected on the Rigaku RU300 rotating anode x-ray generator equipped with focusing x-ray mirrors and operated at 50 kV/100 mA using an R-AXIS IV2+ area detector (Molecular Structure Corporation). The data were integrated with DENZO and scaled with SCALEPACK (19Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326PubMed Google Scholar). All crystals belong to the space group P21212, and have almost identical unit cell parameters to those of the wild-type protein (a = 104.29, b = 95.58, and c = 74.90 Å). The asymmetric unit contains one dimer of the enzyme. The structures of the MAE2 mutants were determined by direct refinement of the structure of the wild type against diffraction data using the program CNS (20Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D. 1998; Scholar). The the of a The refinement for the structures are in and = is the observed of and is over for of = are the observed and structure was with of the bond bond = is the observed of and is over for = are the observed and structure was with of the in a new of the catalytic of the wild-type and the mutant MAE2 enzymes were by the formation of using the method H.M. Kim H.M. Kim Y.S. Biochem. J. Scholar). The for were by the of protein to μl of the which contains of malonamate 16 20 mm and 0.1 m Tris-HCl (pH The was at 37 °C for and the was by μl of the solution containing acid, and 15% enzyme was not the was to 90 was by prior to the at Structure and is a The structures of the two are the we that the absolutely conserved Ser131, and a catalytic triad, and Ser155 is the the (13Shin S. Lee T.H. Ha N.C. Koo H.M. Kim S.Y. Lee H.S. Kim Y.S. Oh B.H. EMBO J. 2002; 21: 2509-2516Google Scholar). The conserved AS the of the and with a of the of the The two most conserved on the AS are and The first is the containing the cis peptide bond between Gly130 and Ser131. The unusual cis configuration two direct between Ser131 and Ser155 The second conserved contains the of the of and is in direct with the two via the with the of Ser131, and The catalytic of the classical Ser-His-Asp triad is known to with the of the serine residue by the histidine residue, which is in the that is to the of the serine residue Structure and W. H. and Scholar). The the base role of the histidine by the of the The same is not for the Ser-cisSer-Lys triad the chemical property of the is different from that of the the of serine is of the Ser-cisSer-Lys triad is most likely in the at and is to function as a base to a from The chemical Ser131 is not to of the of which is from Ser131 the that Ser155 is in the by of the two direct by the and the of and by the by the and the group of (13Shin S. Lee T.H. Ha N.C. Koo H.M. Kim S.Y. Lee H.S. Kim Y.S. Oh B.H. EMBO J. 2002; 21: 2509-2516Google Scholar). the of Ser155 on the of the Ser131 is to function as the catalytic acid that a to the amino which is by the The by the amino group of that the of Ser131. The is in we not other of Ser155 of the triad, the the role of the cis peptide the two of Ser131 to Cysteine the of cysteinyl the serine is with of this family, including human hydrolase EMBO J. 1997; Scholar), protease J. A. J.E. Li M. Wlodawer A. J. Biol. Chem. 2002; Scholar), and B.H. J. J. 1996; Scholar), are known to contain a triad. cysteine protease family members a catalytic triad. The residue of the triad was to for at in T. J. C. Lee A.C. R. J. Biol. Chem. Scholar), which is a of the of cysteine the of to function as a in the active site of MAE2 by Ser155 to the cysteinyl is the serine in the S155C MAE2 mutant was The structure of the mutant enzyme a to this had density from the in the density at The chemical in the of the density indicated that the cysteine residue was as cysteine sulfinic acid, The of this residue with the and the of Ser131. Cysteine sulfinic acid is an of cysteine acid, which is from the of a cysteine residue with a A. Yeh J. D. 1999; Scholar). While cysteine acid is and to to cysteine in the presence of cysteine sulfinic acid is not to the Chem. Res. Scholar, A. Miller H. D. J. Scholar). the enzyme of this mutant was not in the presence of 10 mm we included 1 mm dithiothreitol in all buffer used for the purification of the mutant the cell as well as the mutant enzyme were have been to inside the used for the production of the mutant and the to have the expression of the the active site is of the most conserved peptide both the containing and the containing the similar in all AS family cysteine has not been as the by any AS family enzymes the active site the cysteine residue at this which is and The substitution of the serine of the Ser-His-Asp triad with cysteine results in a in the catalytic of A study that the cysteine is in the and the of the cysteinyl the R.J. Scholar). This was to the structural for the of the The of MAE2 of which appears to an of a to provide a for the of or with the other of the of with to the formation of In the of of two and not the of the cysteine to cysteine acid or the The of structures of the S131A mutant were determined as a complex with the malonamate and with the malonate The of the density of the is from that of the the hydrolysis of the by the mutant enzyme not several of the into the crystals of the mutant enzyme. The mutant about catalytic with the wild-type enzyme. The structure of the mutant that is from the amide nitrogen of the which the mutant to have the The have the The amide of the is in the with the and its amino group is in a bond with the of In the at one of the corresponding to the amide nitrogen of the is and from the of Å). The of the active site are the same in the two the alanine substitution not the of any of the active site to in the structure of the wild-type enzyme in complex with the the in the of the to the S131A mutant and to the wild-type enzyme the In this the amide is from Ser155 and the amide nitrogen of Ser131 which on the basis of the of the structures of the wild-type and the mutant enzyme in complex with the The amide nitrogen to Ser131 Ser155 the and the These structural data suggest that the group of Ser131 is the catalytic acid a to the amino group of the is that the of the S131A mutant is due to the of the catalytic of the the of Ser155 and the of the These are the same catalytic by the histidine of the Ser-His-Asp triad, the of the two as we the residue on the basis of the of Ser131 in the wild-type structure to replace Ser131. The group of the residue with the of and and the of all at of the not These the of the of the active that the substitution of Ser131 with at the structure of the active These observations provide an for the of the serine residue at this of the is conserved in all two AS family which contain the substitution of the glycine residue with alanine or the peptide bond between Gly130 and Ser131 is in the cis we substitution of Gly130 with alanine in the peptide bond between the two with a in the catalytic The structure of the G130A mutant was determined at the of the refinement of the the density for was and that for and Ser131 was that the substitution results in the of this in contrast with the of this in the wild-type enzyme. or the density by a of the peptide bond between and Ser131 in the cis configuration This is for the both MAE2 in the asymmetric unit of the at the of residue in the structure of the wild-type enzyme that its is to the of data not This that has to different from those of Gly130 to the cannot a structure by an The in the mutant enzyme results in in and in the the of the mutant to the cis configuration a in the enzyme The data Gly130 is and the two to the of the glycine residue are the peptide bond was between Gly130 and Ser131, the group of Ser131 cannot a bond with Ser155 and the group of Ser131 cannot the precise and contacts with Ser155 and that are observed for the cis configuration. All observations suggest that the cis configuration of Ser131 is essential for the catalytic function of the triad. and Structural direct of the amino group of and the group of Ser131 that plays the catalytic role of the Ser131 in the of the The structure of the mutant that the lysine residue of the triad plays a structural role as well as the catalytic In the structure of the wild-type the amino group of is in direct with and which are conserved as or with via a a of The substitution of the serine residue with alanine in fatty acid amide hydrolase corresponding to the catalytic by Cravatt B.F. J. Biol. Chem. Scholar), the substitution of the residue with alanine in the peptide amidase corresponding to the catalytic by (14Labahn J. Neumann S. Buldt G. Kula M. Granzin J. J. Mol. Biol. 2002; 322: 1053-1064Google Scholar). The of two in the mutant a of the containing the which several new with and as from the density of the active site Ser155 is completely of from its and with the group of the the is with that in the wild-type enzyme. is to that the of the a of the active site The of the active site the mutant no catalytic In order to the of the bond between and in the structure of the containing the we determined the structure of the T150A mutant enzyme. The structure that two and to in the space by the and the between and not The structure of the of this mutant is the same as that of the wild-type enzyme. the mutant enzyme about of the catalytic of the wild-type enzyme which is similar to the of the corresponding in the peptide amidase (14Labahn J. Neumann S. Buldt G. Kula M. Granzin J. J. Mol. Biol. 2002; 322: 1053-1064Google Scholar). that the that and the via is for the active site the is by the in this In the mutant, the is by the of the of which the direct between and and the of two into the site The of the role of in the active site structure the of the by new of on the catalytic The of the wild-type MAE2 and mutant enzymes are in The of the wild-type enzyme is and that of the S131A mutant is The of the S155C and mutants were In contrast with the of the the substitution of the acid with alanine of the Ser-His-Asp triad not in a of the enzyme of P. Nature. 1988; Scholar). The the catalytic not the of the active site as the mutant a in protease contains a triad, and the of the catalytic of the enzyme are the same as those of trypsin-like P. H. R.J. Gribskov C.L. S. D.L. S.A. C. D.A. Cell. 1996; Scholar). These observations that the acid in the Ser-His-Asp triad plays the catalytic role the lysine residue in the Ser-cisSer-Lys triad plays the structural role as well as the catalytic the which the residue of the novel triad has of other amino acids by the AS family enzymes The study the role of each residue of the Ser-cisSer-Lys triad in and the of the chemical of the triad is absolutely that the triad is not functional the cysteine residue is to cysteine sulfinic The active site of MAE2 appears to the of to cysteine sulfinic The structures of the S131A mutant in complex with the and with the demonstrate that Ser131 is the catalytic acid the amino group of the The formation between the serine and the amino group of the lysine the The cis configuration of Ser131 appears essential for the formation of the two direct with Ser155 and with A modeling that any residue serine at this at the structure of the active The lysine residue was to a structural role of the active site structure as well as the catalytic role of the of the group by Ser131 of the triad. The role appears to by lysine at this that has to precise contacts with the different conserved the structures of AS family enzymes are The AS of the enzymes the catalytic that are similar to each The of the catalytic the triad, and the of the enzymes are with root suggest that the conclusions from this study for the AS family enzymes. The of the Ser-cisSer-Lys triad that the triad is not a variation of the Ser-His-Asp triad or any other known catalytic such as catalytic and that the AS family enzymes are evolutionary distinct from all the other known