Both helical propensity and side-chain hydrophobicity at a partially exposed site in alpha-helix contribute to the thermodynamic stability of ubiquitin

Pfam family DUF1023 consists entirely of uncharacterized proteins generated by sequencing the genomes of Actinobacteria (Bateman A., et al., Nucleic Acids Res. 2004;32 Database issue:D138-141.) Utilizing sequence similarity detection methods, we infer homology between DUF1023 and alpha/beta hydrolases. DUF1023 proteins conserve the core secondary structures in alpha/beta hydrolase fold, and share similar catalytic machinery as that of alpha/beta hydrolases. We predict DUF1023 spatial structure and deduce that they function as hydrolases utilizing catalytic Ser-His-Asp triad with the serine as a nucleophile.     

Enzymatic activity and protein interactions in alpha/beta hydrolase fold proteins: moonlighting versus promiscuity

Genes coding for members of the alpha/beta hydrolase fold superfamily of proteins are present in all known genomes. Although there is no common and essential function performed by these proteins shared in all living organisms, this fold has been used for a number of diverse functions. The ancestry of both enzymatic and protein-protein interaction capability of this structural scaffold made it an important tinkering tool kit for protein function evolution. Recently, enzymes known since a long time have been found to have a second function in acting promiscuously on alternative substrates, or to be true moonlighting proteins acting also as transporters, receptors, chaperones… The reverse situation has been encountered for adhesion proteins shown to be enzymes. This review, while not exhaustive, surveys some of the best-known examples of multiple functions in alpha/beta hydrolase fold proteins.     

Structural comparisons of TIM barrel proteins suggest functional and evolutionary relationships between beta-galactosidase and other glycohydrolases

Beta-galactosidase (lacZ) from Escherichia coli is a 464 kDa homotetramer. Each subunit consists of five domains, the third being an alpha/beta barrel that contains most of the active site residues. A comparison is made between each of the domains and a large set of proteins representative of all structures from the protein data bank. Many structures include an alpha/beta barrel. Those that are most similar to the alpha/beta barrel of E. coli beta-galactosidase have similar catalytic residues and belong to the so-called "4/7 superfamily" of glycosyl hydrolases. The structure comparison suggests that beta-amylase should also be included in this family. Of three structure comparison methods tested, the "ProSup" procedure of Zu-Kang and Sippl and the "Superimpose" procedure of Diederichs were slightly superior in discriminating the members of this superfamily, although all procedures were very powerful in identifying related protein structures. Domains 1, 2, and 4 of E. coli beta-galactosidase have topologies related to "jelly-roll barrels" and "immunoglobulin constant" domains. This fold also occurs in the cellulose binding domains (CBDs) of a number of glycosyl hydrolases. The fold of domain 1 of E. coli beta-galactosidase is closely related to some CBDs, and the domain contributes to substrate binding, but in a manner unrelated to cellulose binding by the CBDs. This is typical of domains 1, 2, 4, and 5, which appear to have been recruited to play roles in beta-galactosidase that are unrelated to the functions that such domains provide in other contexts. It is proposed that beta-galactosidase arose from a prototypical single domain alpha/beta barrel with an extended active site cleft. The subsequent incorporation of elements from other domains could then have reduced the size of the active site from a cleft to a pocket to better hydrolyze the disaccharide lactose and, at the same time, to facilitate the production of inducer, allolactose.     

Threading with chemostructural restrictions method for predicting fold and functionally significant residues: application to dipeptidylpeptidase IV (DPP-IV)

We present a new method for more accurate modeling of protein structure, called threading with chemostructural restrictions. This method addresses those cases in which a target sequence has only remote homologues of known structure for which sequence comparison methods cannot provide accurate alignments. Although remote homologues cannot provide an accurate model for the whole chain, they can be used in constructing practically useful models for the most conserved-and often the most interesting-part of the structure. For many proteins of interest, one can suggest certain chemostructural patterns for the native structure based on the available information on the structural superfamily of the protein, the type of activity, the sequence location of the functionally significant residues, and other factors. We use such patterns to restrict (1) a number of possible templates, and (2) a number of allowed chain conformations on a template. The latter restrictions are imposed in the form of additional template potentials (including terms acting as sequence anchors) that act on certain residues. This approach is tested on remote homologues of alpha/beta-hydrolases that have significant structural similarity in the positions of their catalytic triads. The study shows that, in spite of significant deviations between the model and the native structures, the surroundings of the catalytic triad (positions of C(alpha) atoms of 20-30 nearby residues) can be reproduced with accuracy of 2-3 A. We then apply the approach to predict the structure of dipeptidylpeptidase IV (DPP-IV). Using experimentally available data identifying the catalytic triad residues of DPP-IV (David et al., J Biol Chem 1993;268:17247-17252); we predict a model structure of the catalytic domain of DPP-IV based on the 3D fold of prolyl oligopeptidase (Fulop et al., Cell 1998;94:161-170) and use this structure for modeling the interaction of DPP-IV with inhibitor.     

aCHEdb: the database system for ESTHER, the alpha/beta fold family of proteins and the Cholinesterase gene server.

Acetylcholinesterase belongs to a family of proteins, the alpha/beta hydrolase fold family, whose constituents evolutionarily diverged from a common ancestor and share a similar structure of a central beta sheet surrounded by alpha helices. These proteins fulfil a wide range of physiological functions (hydrolases, adhesion molecules, hormone precursors) [Krejci,E., Duval,N., Chatonnet,A., Vincens,P. and Massoulié,J. (1991) Proc. Natl. Acad. Sci. USA , 88, 6647-6651]. ESTHER (for esterases, alpha/beta hydrolase enzymes and relatives) is a database aimed at collecting in one information system, sequence data together with biological annotations and experimental biochemical results related to the structure-function analysis of the enzymes of the family. The major upgrade of the database comes from the use of a new database management system: aCHEdb which uses the ACeDB program designed by Richard Durbin and Jean Thierry-Mieg. It can be found at http://www.ensam.inra.fr/cholinesterase     

A putative novel alpha/beta hydrolase ORFan family in Bacillus

A large number of sequences in each newly sequenced genome correspond to lineage and species-specific proteins, also known as ORFans. Amongst these ORFans, a large number are sequences with unknown structures and functions. We have identified a family of sequences, annotated as hypothetical proteins, which are specific to Bacillus and have carried out a computational study aimed at characterizing this family. Fold-recognition methods predict that these sequences belong to the alpha/beta hydrolase fold. We suggest possible catalytic triads for the ORFans and propose a hypothesis regarding the possible families within the alpha/beta hydrolase superfamily to which they may belong.     

Diversity and biocatalytic potential of epoxide hydrolases identified by genome analysis

Epoxide hydrolases play an important role in the biodegradation of organic compounds and are potentially useful in enantioselective biocatalysis. An analysis of various genomic databases revealed that about 20% of sequenced organisms contain one or more putative epoxide hydrolase genes. They were found in all domains of life, and many fungi and actinobacteria contain several putative epoxide hydrolase-encoding genes. Multiple sequence alignments of epoxide hydrolases with other known and putative alpha/beta-hydrolase fold enzymes that possess a nucleophilic aspartate revealed that these enzymes can be classified into eight phylogenetic groups that all contain putative epoxide hydrolases. To determine their catalytic activities, 10 putative bacterial epoxide hydrolase genes and 2 known bacterial epoxide hydrolase genes were cloned and overexpressed in Escherichia coli. The production of active enzyme was strongly improved by fusion to the maltose binding protein (MalE), which prevented inclusion body formation and facilitated protein purification. Eight of the 12 fusion proteins were active toward one or more of the 21 epoxides that were tested, and they converted both terminal and nonterminal epoxides. Four of the new epoxide hydrolases showed an uncommon enantiopreference for meso-epoxides and/or terminal aromatic epoxides, which made them suitable for the production of enantiopure (S,S)-diols and (R)-epoxides. The results show that the expression of epoxide hydrolase genes that are detected by analyses of genomic databases is a useful strategy for obtaining new biocatalysts.     

Potential active-site residues in polyneuridine aldehyde esterase, a central enzyme of indole alkaloid biosynthesis, by modelling and site-directed mutagenesis.

In the biosynthesis of the antiarrhythmic alkaloid ajmaline, polyneuridine aldehyde esterase (PNAE) catalyses a central reaction by transforming polyneuridine aldehyde into epi-vellosimine, which is the immediate precursor for the synthesis of the ajmalane skeleton. The PNAE cDNA was previously heterologously expressed in E. coli. Sequence alignments indicated that PNAE has a 43% identity to a hydroxynitrile lyase from Hevea brasiliensis, which is a member of the alpha/beta hydrolase superfamily. The catalytic triad, which is typical for this family, is conserved. By site-directed mutagenesis, the members of the catalytic triad were identified. For further detection of the active residues, a model of PNAE was constructed based on the X-ray crystallographic structure of hydroxynitrile lyase. The potential active site residues were selected on this model, and were mutated in order to better understand the relationship of PNAE with the alpha/beta hydrolases, and as well its mechanism of action. The results showed that PNAE is a novel member of the alpha/beta hydrolase enzyme superfamily.     

Cloning and tissue distribution of three murine alpha/beta hydrolase fold protein cDNAs

We have cloned 3 novel murine cDNAs encoding proteins containing an alpha/beta hydrolase fold; a catalytic domain found in a very wide range of enzymes. These proteins belong to the prosite UPF0017 uncharacterized protein family and we have named them lung alpha/beta hydrolase 1, 2, and 3 (LABH) since they were cloned from lung cDNA. All have 9 coding exons, encoding 412, 425, and 411 residue proteins respectively (46-48 kDa); LABH1 being closely related to LABH3 having 45% identity. All 3 proteins have a single predicted amino-terminus transmembrane domain. An alignment of family members from different phyla enabled the identification of the LABH1 catalytic triad as Ser211, Asp337, and His366. mRNA expression levels of LABH1 and 3 were highest in liver and LABH2 highest in testis. These findings suggest that the LABH proteins consist of a novel family of membrane bound enzymes whose function has yet to be determined.     

An FMN hydrolase of the haloacid dehalogenase superfamily is active in plant chloroplasts

FMN hydrolases catalyze dephosphorylation of FMN to riboflavin. Although these enzymes have been described in many organisms, few had their corresponding genes cloned and their recombinant proteins biochemically characterized, and none had their physiological roles determined. We found previously that FMN hydrolase activity in pea chloroplasts is Mg(2+)-dependent, suggesting an enzyme of the haloacid dehalogenase (HAD) superfamily. In this study, a new FMN hydrolase was purified by multistep chromatography after ammonium sulfate precipitation. The molecular weight of the native protein was estimated at ～59,400, a dimer of about twice the predicted molecular weight of most HAD superfamily phosphatases. After SDS-PAGE of the partially purified material, two separate protein bands within 25-30 kDa were extracted from the gel and analyzed by nanoLC-MS/MS. Peptide sequence matching to the protein samples suggested the presence of three HAD-like hydrolases. cDNAs for sequence homologs from Arabidopsis thaliana of these proteins were expressed in Escherichia coli. Activity screening of the encoded proteins showed that the At1g79790 gene encodes an FMN hydrolase (AtcpFHy1). Plastid localization of AtcpFHy1 was confirmed using fluorescence microscopy of A. thaliana protoplasts transiently expressing the N-terminal fusion of AtcpFHy1 to enhanced green fluorescent protein. Phosphatase activity of AtcpFHy1 is FMN-specific, as assayed with 19 potential substrates. Kinetic parameters and pH and temperature optima for AtcpFHy1 were determined. A phylogenetic analysis of putative phosphatases of the HAD superfamily suggested distinct evolutionary origins for the plastid AtcpFHy1 and the cytosolic FMN hydrolase characterized previously.     

Structure activity studies with xenobiotic substrates using carboxylesterases isolated from Arabidopsis thaliana

Carboxylesterases (CXEs) catalyse the hydrolysis of xenobiotics and natural products radically altering their biological activities. Whereas the substrate selectivity of animal CXEs, such as porcine liver esterase (PLE) have been well studied, the respective enzymes in plants have yet to be defined and their activities determined. Using Arabidopsis thaliana (At) as a source, five representative members of the alpha/beta hydrolase AtCXE family of proteins have been cloned, expressed and the purified recombinant proteins assayed for esterase activity with xenobiotic substrates. Two members, AtCXE5 and AtCXE18 were found to be active carboxylesterases, though AtCXE5 proved to be highly unstable as a soluble protein. AtCXE18 and the previously characterised S-formylglutathione hydrolase from Arabidopsis (AtSFGH) were assayed against a series of esters based on methylumbelliferone in which the acyl moiety was varied with respect to size and conformation. The same series was used to assay crude esterase preparation from Arabidopsis plants and the results compared with those obtained with the commonly used PLE. With straight chain esters, AtCXE18 behaved like PLE, but the Arabidopsis hydrolases proved less tolerant of branched chain acyl components than the mammalian enzyme. While none of the enzyme preparations accurately reflected all the activities determined with crude Arabidopsis protein extracts, the plant enzymes proved more useful than PLE in predicting the hydrolysis of the more sterically constrained esters.     

Dynamics alignment: comparison of protein dynamics in the SCOP database

A novel methodology for comparison of protein dynamics is presented. Protein dynamics is calculated using the Gaussian network model and the modes of motion are globally aligned using the dynamic programming algorithm of Needleman and Wunsch, commonly used for sequence alignment. The alignment is fast and can be used to analyze large sets of proteins. The methodology is applied to the four major classes of the SCOP database: "all alpha proteins," "all beta proteins," "alpha and beta proteins," and "alpha/beta proteins". We show that different domains may have similar global dynamics. In addition, we report that the dynamics of "all alpha proteins" domains are less specific to structural variations within a given fold or superfamily compared with the other classes. We report that domain pairs with the most similar and the least similar global dynamics tend to be of similar length. The significance of the methodology is that it suggests a new and efficient way of mapping between the global structural features of protein families/subfamilies and their encoded dynamics.     

Peptidase family U34 belongs to the superfamily of N-terminal nucleophile hydrolases

Peptidase family U34 consists of enzymes with unclear catalytic mechanism, for instance, dipeptidase A from Lactobacillus helveticus. Using extensive sequence similarity searches, we infer that U34 family members are homologous to penicillin V acylases (PVA) and thus potentially adopt the N-terminal nucleophile (Ntn) hydrolase fold. Comparative sequence and structural analysis reveals a cysteine as the catalytic nucleophile as well as other conserved residues important for catalysis. The PVA/U34 family is variable in sequence and exhibits great diversity in substrate specificity, to include enzymes such as choloyglycine hydrolases, acid ceramidases, isopenicillin N acyltransferases, and a subgroup of eukaryotic proteins with unclear function.     

YbiV from Escherichia coli K12 is a HAD phosphatase

The protein YbiV from Escherichia coli K12 MG1655 is a hypothetical protein with sequence homology to the haloacid dehalogenase (HAD) superfamily of proteins. Although numerous members of this family have been identified, the functions of few are known. Using the crystal structure, sequence analysis, and biochemical assays, we have characterized YbiV as a HAD phosphatase. The crystal structure of YbiV reveals a two-domain protein, one with the characteristic HAD hydrolase fold, the other an inserted alpha/beta fold. In an effort to understand the mechanism, we also solved and report the structures of YbiV in complex with beryllofluoride (BeF3-) and aluminum trifluoride (AlF3), which have been shown to mimic the phosphorylated intermediate and transition state for hydrolysis, respectively, in analogy to other HAD phosphatases. Analysis of the structures reveals the substrate-binding cavity, which is hydrophilic in nature. Both structure and sequence homology indicate YbiV may be a sugar phosphatase, which is supported by biochemical assays that measured the release of free phosphate on a number of sugar-like substrates. We also investigated available genomic and functional data in an effort to determine the physiological substrate.     

Crystal structure of yeast YHR049W/FSH1, a member of the serine hydrolase family

Yhr049w/FSH1 was recently identified in a combined computational and experimental proteomics analysis for the detection of active serine hydrolases in yeast. This analysis suggested that FSH1 might be a serine-type hydrolase belonging to the broad functional alphabeta-hydrolase superfamily. In order to get insight into the molecular function of this gene, it was targeted in our yeast structural genomics project. The crystal structure of the protein confirms that it contains a Ser/His/Asp catalytic triad that is part of a minimal alpha/beta-hydrolase fold. The architecture of the putative active site and analogies with other protein structures suggest that FSH1 may be an esterase. This finding was further strengthened by the unexpected presence of a compound covalently bound to the catalytic serine in the active site. Apparently, the enzyme was trapped with a reactive compound during the purification process.     

Gulosibacter molinativorax ON4T molinate hydrolase, a novel cobalt-dependent amidohydrolase

A new pathway of molinate mineralization has recently been described. Among the five members of the mixed culture able to promote such a process, Gulosibacter molinativorax ON4(T) has been observed to promote the initial breakdown of the herbicide into ethanethiol and azepane-1-carboxylate. In the current study, the gene encoding the enzyme responsible for molinate hydrolysis was identified and heterologously expressed, and the resultant active protein was purified and characterized. Nucleotide sequence analysis revealed that the gene encodes a 465-amino-acid protein of the metal-dependent hydrolase A subfamily of the amidohydrolase superfamily with a predicted molecular mass of 50.9 kDa. Molinate hydrolase shares the highest amino acid sequence identity (48 to 50%) with phenylurea hydrolases of Arthrobacter globiformis and Mycobacterium brisbanense. However, in contrast to previously described members of the metal-dependent hydrolase A subfamily, molinate hydrolase contains cobalt as the only active-site metal.     

Site-directed mutagenesis of potential catalytic residues in 1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase, and hypothesis on the catalytic mechanism of 2,4-dioxygenolytic ring cleavage

1H-3-Hydroxy-4-oxoquinoline 2,4-dioxygenase (Qdo) is a cofactor-free dioxygenase proposed to belong to the alpha/beta hydrolase fold superfamily of enzymes. Alpha/beta Hydrolases contain a highly conserved catalytic triad (nucleophile-acidic residue-histidine). We previously identified a corresponding catalytically essential histidine residue in Qdo. However, as shown by amino acid replacements through site-directed mutagenesis, nucleophilic and acidic residues of Qdo considered as possible triad residues were not absolutely required for activity. This suggests that Qdo does not contain the canonical catalytic triad of the alpha/beta hydrolase fold enzymes. Some radical trapping agents affected the Qdo-catalyzed reaction. A hypothetical mechanism of Qdo-catalyzed dioxygenation of 1H-3-hydroxy-4-oxoquinoline is compared with the dioxygenation of FMNH2 catalyzed by bacterial luciferase, which also uses a histidine residue as catalytic base.     

J1 acylase, a glutaryl-7-aminocephalosporanic acid acylase from Bacillus laterosporus J1, is a member of the alpha/beta-hydrolase fold superfamily

J1 acylase, a glutaryl-7-aminocephalosporanic acid acylase (GCA) isolated from Bacillus laterosporus J1, has been conventionally grouped as the only member of class V GCA, although its amino acid sequence shares less than 10% identity with members of other classes of GCA. Instead, it shows higher sequence similarities with Rhodococcus sp. strain MB1 cocaine esterase (RhCocE) and Acetobacter turbidans alpha-amino acid ester hydrolase (AtAEH), members of the alpha/beta-hydrolase fold superfamily. Homology modeling and secondary structure prediction indicate that the N-terminal region of J1 acylase has an alpha/beta-hydrolase folding pattern. The catalytic triads in RhCocE and AtAEH were identified in J1 acylase as S125, D264 and H309. Mutations to alanine at these positions were found to completely inactivate the enzyme. These results suggest that J1 acylase is a member of the alpha/beta-hydrolase fold superfamily with a serine-histidine-aspartate catalytic triad.     

Mechanistic diversity in a metalloenzyme superfamily

It is now appreciated that the relationships of proteins, particularly enzymes, within a protein superfamily can be understood not only in terms of their sequence similarities and three-dimensional structures but also by chemical threads that relate their functional attributes. The mechanistic ties among superfamily members can often be traced to a common transition state for the rate-limiting step of the reactions being catalyzed. This paper presents an analysis of a metalloenzyme superfamily, the members of which catalyze a very diverse set of reactions with unrelated transition states but a more general common mechanistic imperative. The vicinal oxygen chelate (VOC) superfamily is composed of structurally related proteins with paired beta alpha beta beta beta motifs that provide a metal coordination environment with two or three open or readily accessible coordination sites to promote direct electrophilic participation of the metal ion in catalysis. The known types of reactions that are catalyzed include isomerizations (glyoxalase I), epimerizations (methylmalonyl-CoA epimerase), oxidative cleavage of C-C bonds (extradiol dioxygenase), and nucleophilic substitutions (fosfomycin resistance proteins). The remarkable access to mechanism space that is provided by the VOC superfamily appears to derive from a simple, pseudosymmetric structural fold that maximizes the catalytic versatility of the metal center.     

Crystal structures and proposed structural/functional classification of three protozoan proteins from the isochorismatase superfamily

We have determined the crystal structures of three homologous proteins from the pathogenic protozoans Leishmania donovani, Leishmania major, and Trypanosoma cruzi. We propose that these proteins represent a new subfamily within the isochorismatase superfamily (CDD classification cd004310). Their overall fold and key active site residues are structurally homologous both to the biochemically well-characterized N-carbamoylsarcosine-amidohydrolase, a cysteine hydrolase, and to the phenazine biosynthesis protein PHZD (isochorismase), an aspartyl hydrolase. All three proteins are annotated as mitochondrial-associated ribonuclease Mar1, based on a previous characterization of the homologous protein from L. tarentolae. This would constitute a new enzymatic activity for this structural superfamily, but this is not strongly supported by the observed structures. In these protozoan proteins, the extended active site is formed by inter-subunit association within a tetramer, which implies a distinct evolutionary history and substrate specificity from the previously characterized members of the isochorismatase superfamily. The characterization of the active site is supported crystallographically by the presence of an unidentified ligand bound at the active site cysteine of the T. cruzi structure.