The amidohydrolase superfamily is a functionally diverse group of enzymes found in every organism sequenced to date. The landmark for this superfamily is the conservation of a (beta/alpha)8-barrel structural fold. Isoaspartyl dipeptidase (IAD) from Escherichia coli catalyzes the hydrolytic cleavage of beta-aspartyl dipeptides. Structural studies of the wild-type enzyme demonstrate that the active site consists of a binuclear metal center. Bell-shaped pH-rate profiles are observed for all four metal-substituted forms of the wild-type enzyme and the site-directed mutants, E77Q and Y137F. Structural analysis of IAD with the bound substrate and site-directed mutagenesis shows the importance of the side chains of residues Glu-77, Tyr-137, Arg-169, Arg-233, Asp- 285, and Ser-289 in the substrate binding and hydrolysis. The reaction mechanism for the hydrolysis of dipeptides by IAD is initiated by the polarization of the amide bond via complexation to the beta-metal and the hydrogen bond to Tyr-137. Asp-385 participates in the activation of the bridging hydroxide for nucleophilic attack at the peptide carbon center. The lately protonated Asp-285 donates the proton to the alpha-amino group of the leaving group, causing the collapse of the tetrahedral intermediate and cleavage of the carbon-nitrogen bond. N-formimino-L-glutamate iminohydrolase (HutF) from Pseudomonas aeruginosa acts in the deimination of the fourth intermediate of the histidine degradation pathway, N-formimino-L-glutamate. An amino acid sequence alignment between HutF and other members of the amidohydrolase superfamily containing mononuclear metal centers suggests that the residues Glu-235, His-269, and Asp-320 are involved in substrate binding and deimination. Site-directed mutagenesis of Glu-235, His-269, and Asp-320, in conjunction with the analysis of the four metalsubstituted enzyme forms and pH-rate profiles provides valuable information toward the proposal of a mechanism for deimination of N-formimino-L-glutamate by HutF. This information suggests that the reaction is initiated by the activation of the hydrolytic water through base catalysis via His-269. The enhanced nucleophile attacks the formimino carbon center. In a concerted reaction, Asp-320 deprotonates the hydroxide nucleophile, and His-269 donates a proton to the terminal amino of the iminium group resulting in the collapse of the tetrahedral intermediate, the cleavage of the carbon-nitrogen bond and the release of the products.

The amidohydrolase superfamily is a functionally diverse set of enzymes that catalyzes predominantly hydrolysis reactions involving sugars, nucleic acids, amino acids, and organophosphate esters. A more divergent member of this superfamily, URI (uronate isomerase) from Escherichia coli, catalyzes the isomerization of D-glucuronate to D-fructuronate and D-galacturonate to D-tagaturonate. In Bacillus halodurans, two distinct operons were identified for the metabolism of D-glucuronate and D-galacturonate based on kinetics and genomic context. The canonical uronate isomerase is encoded by the gene Bh0705. A second URI in this organism, Bh0493, is the outlier of the group in terms of sequence similarity. Kinetic evidences indicate that Bh0705 is relatively specific for the isomerization of D-glucuronate, while Bh0493 is specific for the Dgalacturonate pathway. Bell-shaped pH-rate profiles were observed for the wild type URI from Escherichia coli. Primary isotope effects with [2-2H]-D-glucuronate and solvent viscosity studies are consistent with product release as the rate limiting step. X-ray structure of Bh0493 was determined in the presence of D-glucuronate. A chemical mechanism is proposed that utilizes a proton transfer from C-2 of D-glucuronate to C-1 that is initiated by the combined actions of Asp-355 and the C-5 hydroxyl of the substrate that is bound to the metal ion. The formation of the cis-enediol intermediate is further facilitated by the shuttling of the proton between the C-2 and C-1 oxygens by the conserved Tyr-50 and/or Arg-357. Another divergent member of the AHS is the enzyme renal dipeptidase. Structural studies of the enzyme from Streptomyces coelicolor (Sco3058) demonstrate that the active site consists of a binuclear metal center. Bell-shaped pH-rate profiles are observed for both Zn2+ and Cd2+ enzymes. A chemical mechanism for renal dipeptidase is proposed based on structural analysis of the enzyme-inhibitor complex. The reaction is initiated by the polarization of the amide bond by the B-metal. Asp-320 activates the bridging hydroxide for nucleophilic attack at the peptide carbon center, forming a tetrahedral intermediate that is stabilized by the metal center and His-150. The protonated Asp-320 donates the proton to the a-amino group of the leaving group, causing the collapse of the tetrahedral intermediate and cleavage of the carbon-nitrogen bond.

The Amidohydrolase Superfamily (AHS) of enzymes is one of the most structurally and functionally studied groups of biological catalysts, exquisitely designed to carry out an extensive number of reactions defined by a similar reaction mechanism. There are approximately 11,000 genes coding for AHS proteins from about 2,100 sequenced organisms. Sequence information for these genes has been catalogued in databases, the most instrumental being the National Center for Biotechnology Information (NCBI). Despite the accessible information organized in genomic databases, there is still an extensive problem of reliability in the functional annotation of gene products assigned to the AHS. Proteins in COG3964 of the AHS have been functionally identified as dihydroorotases and adenine deaminases. Eight proteins within three group families of COG3964 have been purified and fail to demonstrate the functionally annotated activity. A library of compounds developed by functional-group modifications was compiled and tested with these enzymes. A group of enzymes within COG3964 demonstrates the ability to hydrolyze stereospecific acetylated alpha-hydroxyl carboxylates. Substrate profiles were constructed for enzymes belonging to group 6 of COG3964. Atu3266, Oant2987 and RHE_PE00295 hydrolyze the R-isomers of a library of alpha-acetyl carboxylates of which acetyl-R-mandelate is the best substrate with catalytic efficiencies of 10^5 M^-1s^-1. This compound was identified after a series of modifications from a low-activity compound (V/K = 4 M^-1s^-1). Methylphosphonate analogs of acetyl-R-mandelate and N-acetyl-D-phenyl glycine are inhibitors of enzymes in group 6. The structure of Atu3266 was used in docking experiments to assess the selectivity of R- enantiomers over their S- counterparts. An additional group of orthologues share less than 40% sequence similarity to enzymes from group 6. EF0837, STM4445 and BCE_5003 from group 2 show significantly lower rates for the hydrolysis of alpha-acetyl carboxylates, including acetyl-R-mandelate, hydrolyzed at values of kcat/Km = 10^3 M^-1s^-1. This is also the only active compound for EF0837. Xaut_0650 and blr3349 from group 7 of COG3964 demonstrate less than 30% identity to enzymes in groups 2 and 6. These enzymes fail to hydrolyze any compound from an extended library of compounds. An annotated selenocysteine synthase gene (SelA) from COG1921 has been identified as a gene neighbor to almost every amidohydrolase from COG3964. Atu3263, Oant2990 and EF0838 are pyridoxal-5’-phosphate dependent enzymes that were purified and assayed with D- and L- amino acids. Initial thermal-shift fluorescence assays determined that in the presence of D-cysteine, the proteins were denatured at lower temperatures.

Phosphotriesterase (PTE) from Pseudomonas diminuta is a zinc metalloenzyme found in soil bacteria capable of organophosphate hydrolysis at rates approaching the diffusion controlled limit. Interest in PTE for degradation of chemical warfare agents and disposal of pesticides supports the need to understand the mechanism by which it performs hydrolysis. For further mechanistic clarity, this work will provide direct confirmation of the solvent bridge identity and the protonated species resulting in loss of catalytic identity. Inhibitor and product binding to the metal center will also be addressed; as well as the evaluation of the catalytic activity of Fe(II)-substituted PTE. This work has determined that the Mn/Mn-PTE electron paramagnetic resonance (EPR) spectrum exhibits exchange coupling that is facilitated through a hydroxide bridge. Protonation of the bridging hydroxide results in the loss of the exchange coupling between the two divalent cations and the loss of catalytic activity. The reversible protonation of the bridging hydroxide has an apparent pKa of 7.3 based upon changes in the EPR spectrum of Mn/Mn-PTE with alterations in pH. The pH-rate profile for the hydrolysis of paraoxon by Mn/Mn-PTE shows the requirement of a single function group that must be unprotonated with a pKa of 7.1. The comparable pKa values are proposed to result from the protonation of the same ionizable species. The effects of inhibitor and product binding on the magnetic properties of the metal center and the hydroxyl bridge are investigated by accessing new EPR spectral features. This work concludes that the binding of inhibitor occurs at the metal center and results in an increase of non-bridged hydroxyl species. These results, in conjunction with kinetic and crystallographic data, suggest that substrate binding via the phosphoryl oxygen at the ?-metal weakens the hydroxyl bridge coordination to the ?-metal. This loss of coordination would increase the nucleophilic character of the bridge, and binding of the substrate to the metal center would result in a stronger nucleophile for hydrolysis. Lastly, Fe(II) binding and activation of apoenzyme is evaluated under anaerobic conditions. This work concludes Fe/Fe-PTE is not catalytically active, but can bind up to 2 equivalent Fe(II) ions per active site.

Improved sequencing technologies have created an explosion of sequence information that is analyzed and proteins are annotated automatically. Annotations are made based on similarity scores to previously annotated sequences, so one misannotation is propagated throughout databases and the number of misannotated proteins grows with the number of sequenced genomes. A systematic approach to correctly identify the function of proteins in the amidohydrolase superfamily is described in this work using Clusters of Orthologous Groups of proteins as defined by NCBI. The focus of this work is COG1816, which contains proteins annotated, often incorrectly, as adenosine deaminase enzymes. Sequence similarity networks were used to evaluate the relationship between proteins. Proteins previously annotated as adenosine deaminases: Pa0148 (Pseudomonas aeruginosa PAO1), AAur_1117 (Arthrobacter aurescens TC1), Sgx9403e and Sgx9403g, were purified and their substrate profiles revealed that adenine and not adenosine was a substrate for these enzymes. All of these proteins will deaminate adenine with values of kcat/Km that exceed 105 M-1s-1. A small group of enzymes similar to Pa0148 was discovered to catalyze the hydrolysis of N-6-substituted adenine derivatives, several of which are cytokinins, a common type of plant hormone. Patl2390, from Pseudoalteromonas atlantica T6c, was shown to hydrolytically deaminate N-6-isopentenyladenine to hypoxanthine and isopentenylamine with a kcat/Km of 1.2 x 107 M^-1 s^-1. This enzyme does not catalyze the deamination of adenine or adenosine. Two small groups of proteins from COG1816 were found to have 6-aminodeoxyfutalosine as their true substrate. This function is shared with 2 small groups of proteins closely related to guanine and cytosine deaminase from COG0402. The deamination of 6-aminofutalosine is part of the alternative menaquinone biosynthetic pathway that involves the formation of futalosine. 6-Aminofutalosine is deaminated with a catalytic effeciency of 105 M-1s-1 or greater, Km’s of 0.9 to 6.0 µM and kcat’s of 1.2 to 8.6 s-1. Another group of proteins was shown to deaminate cyclic- 3’, 5’ -adenosine monophosphate (cAMP) to produce cyclic-3’, 5’-inosine monophosphate, but will not deaminate adenosine, adenine or adenosine monophosphate. This protein was cloned from a human pathogen, Leptospira interrogans. Deamination may function in regulating the signaling activities of cAMP.

N-Acylethanolamines (NAEs) are endogenous lipid metabolites that occur in a variety of dry seeds, and their levels decline rapidly during the first few hours of imbibition (Chapman et al., 1999, Plant Physiol., 120:1157-1164). Biochemical studies supported the existence of an NAE amidohydrolase activity in seeds and seedlings, and efforts were directed toward identification of DNA sequences encoding this enzyme. Mammalian tissues metabolize NAEs via an amidase enzyme designated fatty acid amide hydrolase (FAAH). Based on the characteristic amidase signature sequence in mammalian FAAH, a candidate Arabidopsis cDNA was identified and isolated by reverse transcriptase-PCR. The Arabidopsis cDNA was expressed in E. coli and the recombinant protein indeed hydrolyzed a range of NAEs to free fatty acids and ethanolamine. Kinetic parameters for the recombinant protein were consistent with those properties of the rat FAAH, supporting identification of this Arabidopsis cDNA as a FAAH homologue. Two T-DNA insertional mutant lines with disruptions in the Arabidopsis NAE amidohydrolase gene (At5g64440) were identified. The homozygous mutant seedlings were more sensitive than the wild type to exogenously applied NAE 12:0. Transgenic seedlings overexpressing the NAE amidohydrolase enzyme showed noticeably greater tolerance to NAE 12:0 than wild type seedlings. These results together provide evidence in vitro and in vivo for the molecular identification of Arabidopsis NAE amidohydrolase. Moreover, the plants with altered NAE amidohydrolase expression may provide new tools for improved understanding of the role of NAEs in germination and seedling growth.

J'ai mis au point une méthodologie pour annoter les superfamilles d'enzymes, en décrire l'histoire et les replacer dans l'évolution de leurs voies métaboliques. J'en ai étudié trois : (1) les amidohydrolases cycliques, dont les DHOases (dihydroorotases, biosynthèse des pyrimidines), pour lesquelles j'ai proposé une nouvelle classification. L'arbre phylogénétique inclut les dihydropyrimidinases (DHPases) et allantoïnases (ALNases) qui ont des réactions similaires dans d'autres voies (dégradation des pyrimidines et des purines respectivement). (2) L'étude de la superfamille des DHODases (qui suivent les DHOases) montre une phylogénie semblable aux DHOases, avec également des enzymes d'autres voies, dont les DHPDases (qui suivent les DHPases). De cette observation est né le concept de module réactionnel, qui correspond à la conservation de l’enchaînement de réactions semblables dans différentes voies métaboliques. Cela a été utilisé lors de (3) l'étude des carbamoyltransférases (TCases) qui incluent les ATCases (précédant les DHOases). J'ai d'abord montré l'existence d'une nouvelle TCase potentiellement impliquée dans la dégradation des purines et lui ai proposé un nouveau rôle en utilisant le concept de module réactionnel (enchaînement avec l'ALNase). Dans ces trois grandes familles j'ai aussi mis en évidence trois groupes de paralogues non identifiés qui se retrouvent pourtant dans un même contexte génétique appelé « Yge » et qui formeraient donc un module réactionnel constitutif d'une nouvelle voie hypothétique. Appliqué à diverses voies, le concept de modules réactionnels refléterait donc les voies métaboliques ancestrales dont ils seraient les éléments de base. / I designed a methodology to annotate enzyme superfamilies, explain their history and describe them in the context of metabolic pathways evolution. Three superfamilies were studied: (1) cyclic amidohydrolases, including DHOases (dihydroorotases, third step of the pyrimidines biosynthesis), for which I proposed a new classification. The phylogenetic tree also includes dihydropyrimidinases (DHPases) and allantoinases (ALNases) which catalyze similar reactions in other pathways (pyrimidine and purine degradation, respectively). (2) The DHODases superfamily (after DHOases) show a similar phylogeny as DHOases, including enzymes from other pathways, DHPDases in particular (after DHPases). This led to the concept of reaction module, i.e. a conserved series of similar reactions in different metabolic pathways. This was used to study (3) the carbamoyltransferases (TCases) which include ATCases (before DHOases). I first isolated a new kind of TCase, potentially involved in the purine degradation, and I proposed a new role for it in the light of reaction modules (linked with ALNase). In those three superfamilies I also found three groups of unidentified paralogs that were remarkably part of the same genetic context called “Yge” which would be a reaction module part of an unidentified pathway. The concept of reactions modules may then reflect the ancestral metabolic pathways for which they would be basic elements.

α-Amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD) and α-aminomuconate-ε-semialdehyde dehydrogenase (AMSDH) are two neighboring enzymes in the L-tryptophan and 2-nitrobenzoic acid degradation pathways. The substrates of the two enzymes, α-amino-β-carboxymuconate-ε-semialdehyde (ACMS) and α-aminomuconate-ε-semialdehyde (2-AMS), are unstable and spontaneously decay to quinolinic acid and picolinic acid, respectively. ACMSD utilizes a divalent zinc metal as cofactor and is a member of the amidohydrolase superfamily. In this dissertation work, we have identified an important histidine residue in the active site that plays dual roles in tuning metal selectivity and activating a metal bound water ligand using mutagenesis, resonance Raman, EPR, crystallography, and ICP metal analysis techniques. The crystal structures of ACMSD from Pseudomonas fluorescens (PfACMSD) have been solved as homodimers in our laboratory while human ACMSD (hACMSD) was annotated as a monomer by another group. To resolve this structural difference, we used two conserved active site arginine residues as probes to study the oligomeriztion state of ACMSD and demonstrated that these two arginine residues are involved in substrate binding and that both Pf- and h- ACMSD are catalytically active only in the dimeric state. Subsequently, we solved the crystal structure of hACMSD and found it to be a homodimer in both catalytically active and inhibitor-bound forms. AMSDH is an NAD+ dependent enzyme and belongs to the aldehyde dehydrogenase superfamily. Due to the high instability of its substrate, AMSDH has not been studied at the molecular level prior to our work. We have cloned and expressed PfAMSDH in E. coli. The purified protein has high activity towards both 2-AMS and 2-hydroxymuconate semialdehyde (2-HMS), a stable substrate analog. We have successfully crystallized AMSDH with/without NAD+ and solved the crystal structure at up to 1.95 Å resolution. Substrate bound ternary complex structures were obtained by soaking the NAD+ containing crystals with 2-AMS or 2-HMS. Notably, two covalently bound catalytic intermediates were captured and characterized using a combination of crystallography, stopped-flow, single crystal spectroscopy, and mass spectrometry. The first catalytic working model of AMSDH has been proposed based on our success in structural and spectroscopic characterization of the enzyme in five catalytically relevant states in this dissertation work.

The amidohydrolase superfamily is a functionally diverse group of evolutionarily related proteins which utilize metal cofactors in the activation of a hydrolytic water molecule and in the stabilization of the resulting tetrahedral intermediate. Members of this superfamily have been described which use one or two divalent transition metals. These metal cofactors are located in either or both of two active-site metal binding centers which are labeled as the Ma and MB sites. The goal of this research was to elucidate the nature of the reactions catalyzed by Ma and MB mononuclear members of the amidohydrolase superfamily. This was approached through comprehensive mechanistic evaluations of two enzymes which utilized the different metal sites. Nacetyl- D-glucosamine-6-phosphate deacetylase from E. coli (NagA) and cytosine deaminase from E. coli (CDA) served as models for mononuclear amidohydrolase superfamily enzymes which have evolved to utilize a single B-metal and a single a-metal for hydrolysis, respectively. This research elucidated the different properties imparted by the distinct a and B active sites and the specific interactions utilized by the enzymes for substrate binding and catalysis. These studies led to the eventual proposal of detailed chemical mechanisms and the identification of rate determining steps. Knowledge of sequence-function relationships was applied toward the discovery of function for enzymes related to cytosine deaminase and guanine deaminase. The first group of enzymes investigated was proposed to catalyze the fourth step in riboflavin and coenzyme F420 biosynthesis in Achaea. Three putative deaminases; Mm0823 from Methanosarcina mazei, MmarC7_0625 from Methanococcus maripaludis C7 and Sso0398 from Sulfolobus solfataricus were cloned and expressed. These proteins proved to be intractably insoluble. A second set of enzymes, Pa0142 from Pseudomonas aeruginosa PA01 and SGX-9236e (with crystal structure PDB: 3HPA) were found to catalyze the novel deamination of 8-oxoguanine, a mutagenic product of DNA oxidation. 9236e was cloned from an unidentified environmental sample of the Sargasso Sea. The closest homolog (98% identical) is Bcep18194_A5267 from Burkholderia sp. 383. Additionally, it was discovered that the proteins SGX-9339a (with crystal structure PDB: 2PAJ) and SGX-9236b catalyzed the deamination of isoxanthopterin and pterin-6- carboxylate in a poorly characterized folate degradation pathway. These enzymes were also from unknown environmental samples of the Sargasso Sea. The closest homolog of 9339a (88% identical) is Bxe_A2016 from Burkholderia xenovorans LB400. The closest homolog of 9236b (95% identical) is Bphyt_7136 from Burkholderia phytofirmans PsJN.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.