An essential bifunctional enzyme in Mycobacterium tuberculosis for itaconate dissimilation and leucine catabolism.

Mycobacterium tuberculosis (Mtb) is the etiological agent of tuberculosis. One-fourth of the global population is estimated to be infected with Mtb, accounting for ∼1.3 million deaths in 2017. As part of the immune response to Mtb infection, macrophages produce metabolites with the purpose of inhibiting or killing the bacterial cell. Itaconate is an abundant host metabolite thought to be both an antimicrobial agent and a modulator of the host inflammatory response. However, the exact mode of action of itaconate remains unclear. Here, we show that Mtb has an itaconate dissimilation pathway and that the last enzyme in this pathway, Rv2498c, also participates in l-leucine catabolism. Our results from phylogenetic analysis, in vitro enzymatic assays, X-ray crystallography, and in vivo Mtb experiments, identified Mtb Rv2498c as a bifunctional ß-hydroxyacyl-CoA lyase and that deletion of the rv2498c gene from the Mtb genome resulted in attenuation in a mouse infection model. Altogether, this report describes an itaconate resistance mechanism in Mtb and an l-leucine catabolic pathway that proceeds via an unprecedented (R)-3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) stereospecific route in nature.

Luminescent Carbon Dots from Wet Olive Pomace: Structural Insights, Photophysical Properties and Cytotoxicity.

Carbon nanomaterials endowed with significant luminescence have been synthesized for the first time from an abundant, highly localized waste, the wet pomace (WP), a semi-solid by-product of industrial olive oil production. Synthetic efforts were undertaken to outshine the photoluminescence (PL) of carbon nanoparticles through a systematic search of the best reaction conditions to convert the waste biomass, mainly consisting in holocellulose, lignin and proteins, into carbon dots (CDs) by hydrothermal carbonization processes. Blue-emitting CDs with high fluorescence quantum yields were obtained. Using a comprehensive set of spectroscopic tools (FTIR, Raman, XPS, and 1H/13C NMR) in combination with steady-state and time-resolved fluorescence spectroscopy, a rational depiction of WP-CDs structures and their PL properties was reached. WP-CDs show the up-conversion of PL capabilities and negligible cytotoxicity against two mammalian cell lines (L929 and HeLa). Both properties are excellent indicators for their prospective application in biological imaging, biosensing, and dynamic therapies driven by light.

Anti-CTLA-4 therapy requires an Fc domain for efficacy.

Ipilimumab, a monoclonal antibody that recognizes cytotoxic T lymphocyte antigen (CTLA)-4, was the first approved "checkpoint"-blocking anticancer therapy. In mouse tumor models, the response to antibodies against CTLA-4 depends entirely on expression of the Fcγ receptor (FcγR), which may facilitate antibody-dependent cellular phagocytosis, but the contribution of simple CTLA-4 blockade remains unknown. To understand the role of CTLA-4 blockade in the complete absence of Fc-dependent functions, we developed H11, a high-affinity alpaca heavy chain-only antibody fragment (VHH) against CTLA-4. The VHH H11 lacks an Fc portion, binds monovalently to CTLA-4, and inhibits interactions between CTLA-4 and its ligand by occluding the ligand-binding motif on CTLA-4 as shown crystallographically. We used H11 to visualize CTLA-4 expression in vivo using whole-animal immuno-PET, finding that surface-accessible CTLA-4 is largely confined to the tumor microenvironment. Despite this, H11-mediated CTLA-4 blockade has minimal effects on antitumor responses. Installation of the murine IgG2a constant region on H11 dramatically enhances its antitumor response. Coadministration of the monovalent H11 VHH blocks the efficacy of a full-sized therapeutic antibody. We were thus able to demonstrate that CTLA-4-binding antibodies require an Fc domain for antitumor effect.

Monitoring the Cortical Activity of Children and Adults during Cognitive Task Completion.

In this paper, we used an EEG system to monitor and analyze the cortical activity of children and adults at a sensor level during cognitive tasks in the form of a Schulte table. This complex cognitive task simultaneously involves several cognitive processes and systems: visual search, working memory, and mental arithmetic. We revealed that adults found numbers on average two times faster than children in the beginning. However, this difference diminished at the end of table completion to 1.8 times. In children, the EEG analysis revealed high parietal alpha-band power at the end of the task. This indicates the shift from procedural strategy to less demanding fact-retrieval. In adults, the frontal beta-band power increased at the end of the task. It reflects enhanced reliance on the top-down mechanisms, cognitive control, or attentional modulation rather than a change in arithmetic strategy. Finally, the alpha-band power of adults exceeded one of the children in the left hemisphere, providing potential evidence for the fact-retrieval strategy. Since the completion of the Schulte table involves a whole set of elementary cognitive functions, the obtained results were essential for developing passive brain-computer interfaces for monitoring and adjusting a human state in the process of learning and solving cognitive tasks of various types.

Investigating the role of a backbone to substrate hydrogen bond in OMP decarboxylase using a site-specific amide to ester substitution.

Hydrogen bonds between backbone amide groups of enzymes and their substrates are often observed, but their importance in substrate binding and/or catalysis is not easy to investigate experimentally. We describe the generation and kinetic characterization of a backbone amide to ester substitution in the orotidine 5'-monophosphate (OMP) decarboxylase from Methanobacter thermoautotrophicum (MtOMPDC) to determine the importance of a backbone amide-substrate hydrogen bond. The MtOMPDC-catalyzed reaction is characterized by a rate enhancement (∼10(17)) that is among the largest for enzyme-catalyzed reactions. The reaction proceeds through a vinyl anion intermediate that may be stabilized by hydrogen bonding interaction between the backbone amide of a conserved active site serine residue (Ser-127) and oxygen (O4) of the pyrimidine moiety and/or electrostatic interactions with the conserved general acidic lysine (Lys-72). In vitro translation in conjunction with amber suppression using an orthogonal amber tRNA charged with L-glycerate ((HO)S) was used to generate the ester backbone substitution (S127(HO)S). With 5-fluoro OMP (FOMP) as substrate, the amide to ester substitution increased the value of Km by ∼1.5-fold and decreased the value of kcat by ∼50-fold. We conclude that (i) the hydrogen bond between the backbone amide of Ser-127 and O4 of the pyrimidine moiety contributes a modest factor (∼10(2)) to the 10(17) rate enhancement and (ii) the stabilization of the anionic intermediate is accomplished by electrostatic interactions, including its proximity of Lys-72. These conclusions are in good agreement with predictions obtained from hybrid quantum mechanical/molecular mechanical calculations.

Loss of quaternary structure is associated with rapid sequence divergence in the OSBS family.

The rate of protein evolution is determined by a combination of selective pressure on protein function and biophysical constraints on protein folding and structure. Determining the relative contributions of these properties is an unsolved problem in molecular evolution with broad implications for protein engineering and function prediction. As a case study, we examined the structural divergence of the rapidly evolving o-succinylbenzoate synthase (OSBS) family, which catalyzes a step in menaquinone synthesis in diverse microorganisms and plants. On average, the OSBS family is much more divergent than other protein families from the same set of species, with the most divergent family members sharing <15% sequence identity. Comparing 11 representative structures revealed that loss of quaternary structure and large deletions or insertions are associated with the family's rapid evolution. Neither of these properties has been investigated in previous studies to identify factors that affect the rate of protein evolution. Intriguingly, one subfamily retained a multimeric quaternary structure and has small insertions and deletions compared with related enzymes that catalyze diverse reactions. Many proteins in this subfamily catalyze both OSBS and N-succinylamino acid racemization (NSAR). Retention of ancestral structural characteristics in the NSAR/OSBS subfamily suggests that the rate of protein evolution is not proportional to the capacity to evolve new protein functions. Instead, structural features that are conserved among proteins with diverse functions might contribute to the evolution of new functions.

Substrate Distortion and the Catalytic Reaction Mechanism of 5-Carboxyvanillate Decarboxylase.

5-Carboxyvanillate decarboxylase (LigW) catalyzes the conversion of 5-carboxyvanillate to vanillate in the biochemical pathway for the degradation of lignin. This enzyme was shown to require Mn(2+) for catalytic activity and the kinetic constants for the decarboxylation of 5-carboxyvanillate by the enzymes from Sphingomonas paucimobilis SYK-6 (kcat = 2.2 s(-1) and kcat/Km = 4.0 × 10(4) M(-1) s(-1)) and Novosphingobium aromaticivorans (kcat = 27 s(-1) and kcat/Km = 1.1 × 10(5) M(-1) s(-1)) were determined. The three-dimensional structures of both enzymes were determined in the presence and absence of ligands bound in the active site. The structure of LigW from N. aromaticivorans, bound with the substrate analogue, 5-nitrovanillate (Kd = 5.0 nM), was determined to a resolution of 1.07 Å. The structure of this complex shows a remarkable enzyme-induced distortion of the nitro-substituent out of the plane of the phenyl ring by approximately 23°. A chemical reaction mechanism for the decarboxylation of 5-carboxyvanillate by LigW was proposed on the basis of the high resolution X-ray structures determined in the presence ligands bound in the active site, mutation of active site residues, and the magnitude of the product isotope effect determined in a mixture of H2O and D2O. In the proposed reaction mechanism the enzyme facilitates the transfer of a proton to C5 of the substrate prior to the decarboxylation step.

Structure of N-formimino-L-glutamate iminohydrolase from Pseudomonas aeruginosa.

N-Formimino-l-glutamate iminohydrolase (HutF), from Pseudomonas aeruginosa with a locus tag of Pa5106 ( gi|15600299 ), is a member of the amidohydrolase superfamily. This enzyme catalyzes the deamination of N-formimino-l-glutamate to N-formyl-l-glutamate and ammonia in the histidine degradation pathway. The crystal structure of Pa5106 was determined in the presence of the inhibitors N-formimino-l-aspartate and N-guanidino-l-glutaric acid at resolutions of 1.9 and 1.4 Å, respectively. The structure of an individual subunit is composed of two domains with the larger domain folding as a distorted (ß/α)8-barrel. The (ß/α)8-barrel domain is composed of eight ß-strands flanked by 11 α-helices, whereas the smaller domain is made up of eight ß-strands. The active site of Pa5106 contains a single zinc atom that is coordinated by His-56, His-58, His-232, and Asp-320. The nucleophilic solvent water molecule coordinates with the zinc atom at a distance of 2.0 Å and is hydrogen bonded to Asp-320 and His-269. The α-carboxylate groups of both inhibitors are hydrogen bonded to the imidazole moiety of His-206, the hydroxyl group of Tyr-121, and the side chain amide group of Gln-61. The side chain carboxylate groups of the two inhibitors are ion-paired with the guanidino groups of Arg-209 and Arg-82. Computational docking of high-energy tetrahedral intermediate forms of the substrate, N-formimino-l-glutamate, to the three-dimensional structure of Pa5106 suggests that this compound likely undergoes a re-faced nucleophilic attack at the formimino group by the metal-bound hydroxide. A catalytic mechanism of the reaction catalyzed by Pa5106 is proposed.

Homology models guide discovery of diverse enzyme specificities among dipeptide epimerases in the enolase superfamily.

The rapid advance in genome sequencing presents substantial challenges for protein functional assignment, with half or more of new protein sequences inferred from these genomes having uncertain assignments. The assignment of enzyme function in functionally diverse superfamilies represents a particular challenge, which we address through a combination of computational predictions, enzymology, and structural biology. Here we describe the results of a focused investigation of a group of enzymes in the enolase superfamily that are involved in epimerizing dipeptides. The first members of this group to be functionally characterized were Ala-Glu epimerases in Eschericiha coli and Bacillus subtilis, based on the operon context and enzymological studies; these enzymes are presumed to be involved in peptidoglycan recycling. We have subsequently studied more than 65 related enzymes by computational methods, including homology modeling and metabolite docking, which suggested that many would have divergent specificities;, i.e., they are likely to have different (unknown) biological roles. In addition to the Ala-Phe epimerase specificity reported previously, we describe the prediction and experimental verification of: (i) a new group of presumed Ala-Glu epimerases; (ii) several enzymes with specificity for hydrophobic dipeptides, including one from Cytophaga hutchinsonii that epimerizes D-Ala-D-Ala; and (iii) a small group of enzymes that epimerize cationic dipeptides. Crystal structures for certain of these enzymes further elucidate the structural basis of the specificities. The results highlight the potential of computational methods to guide experimental characterization of enzymes in an automated, large-scale fashion.

Discovery of a bacterial 5-methylcytosine deaminase.

5-Methylcytosine is found in all domains of life, but the bacterial cytosine deaminase from Escherichia coli (CodA) will not accept 5-methylcytosine as a substrate. Since significant amounts of 5-methylcytosine are produced in both prokaryotes and eukaryotes, this compound must eventually be catabolized and the fragments recycled by enzymes that have yet to be identified. We therefore initiated a comprehensive phylogenetic screen for enzymes that may be capable of deaminating 5-methylcytosine to thymine. From a systematic analysis of sequence homologues of CodA from thousands of bacterial species, we identified putative cytosine deaminases where a "discriminating" residue in the active site, corresponding to Asp-314 in CodA from E. coli, was no longer conserved. Representative examples from Klebsiella pneumoniae (locus tag: Kpn00632), Rhodobacter sphaeroides (locus tag: Rsp0341), and Corynebacterium glutamicum (locus tag: NCgl0075) were demonstrated to efficiently deaminate 5-methylcytosine to thymine with values of kcat/Km of 1.4 × 10(5), 2.9 × 10(4), and 1.1 × 10(3) M(-1) s(-1), respectively. These three enzymes also catalyze the deamination of 5-fluorocytosine to 5-fluorouracil with values of kcat/Km of 1.2 × 10(5), 6.8 × 10(4), and 2.0 × 10(2) M(-1) s(-1), respectively. The three-dimensional structure of Kpn00632 was determined by X-ray diffraction methods with 5-methylcytosine (PDB id: 4R85 ), 5-fluorocytosine (PDB id: 4R88 ), and phosphonocytosine (PDB id: 4R7W ) bound in the active site. When thymine auxotrophs of E. coli express these enzymes, they are capable of growth in media lacking thymine when supplemented with 5-methylcytosine. Expression of these enzymes in E. coli is toxic in the presence of 5-fluorocytosine, due to the efficient transformation to 5-fluorouracil.

Discovery of function in the enolase superfamily: D-mannonate and d-gluconate dehydratases in the D-mannonate dehydratase subgroup.

The continued increase in the size of the protein sequence databases as a result of advances in genome sequencing technology is overwhelming the ability to perform experimental characterization of function. Consequently, functions are assigned to the vast majority of proteins via automated, homology-based methods, with the result that as many as 50% are incorrectly annotated or unannotated ( Schnoes et al. PLoS Comput. Biol. 2009 , 5 ( 12 ), e1000605 ). This manuscript describes a study of the D-mannonate dehydratase (ManD) subgroup of the enolase superfamily (ENS) to investigate how function diverges as sequence diverges. Previously, one member of the subgroup had been experimentally characterized as ManD [dehydration of D-mannonate to 2-keto-3-deoxy-D-mannonate (equivalently, 2-keto-3-deoxy-D-gluconate)]. In this study, 42 additional members were characterized to sample sequence-function space in the ManD subgroup. These were found to differ in both catalytic efficiency and substrate specificity: (1) high efficiency (kcat/KM = 10(3) to 10(4) M(-1) s(-1)) for dehydration of D-mannonate, (2) low efficiency (kcat/KM = 10(1) to 10(2) M(-1) s(-1)) for dehydration of d-mannonate and/or D-gluconate, and 3) no-activity with either D-mannonate or D-gluconate (or any other acid sugar tested). Thus, the ManD subgroup is not isofunctional and includes D-gluconate dehydratases (GlcDs) that are divergent from the GlcDs that have been characterized in the mandelate racemase subgroup of the ENS (Lamble et al. FEBS Lett. 2004 , 576 , 133 - 136 ) (Ahmed et al. Biochem. J. 2005 , 390 , 529 - 540 ). These observations signal caution for functional assignment based on sequence homology and lay the foundation for the studies of the physiological functions of the GlcDs and the promiscuous ManDs/GlcDs.

Computational, structural, and kinetic evidence that Vibrio vulnificus FrsA is not a cofactor-independent pyruvate decarboxylase.

The fermentation-respiration switch (FrsA) protein in Vibrio vulnificus was recently reported to catalyze the cofactor-independent decarboxylation of pyruvate. We now report quantum mechanical/molecular mechenical calculations that examine the energetics of C-C bond cleavage for a pyruvate molecule bound within the putative active site of FrsA. These calculations suggest that the barrier to C-C bond cleavage in the bound substrate is 28 kcal/mol, which is similar to that estimated for the uncatalyzed decarboxylation of pyruvate in water at 25 °C. In agreement with the theoretical predictions, no pyruvate decarboxylase activity was detected for recombinant FrsA protein that could be crystallized and structurally characterized. These results suggest that the functional annotation of FrsA as a cofactor-independent pyruvate decarboxylase is incorrect.

Structural and mechanistic characterization of L-histidinol phosphate phosphatase from the polymerase and histidinol phosphatase family of proteins.

L-Histidinol phosphate phosphatase (HPP) catalyzes the hydrolysis of L-histidinol phosphate to L-histidinol and inorganic phosphate, the penultimate step in the biosynthesis of L-histidine. HPP from the polymerase and histidinol phosphatase (PHP) family of proteins possesses a trinuclear active site and a distorted (ß/α)(7)-barrel protein fold. This group of enzymes is closely related to the amidohydrolase superfamily of enzymes. The mechanism of phosphomonoester bond hydrolysis by the PHP family of HPP enzymes was addressed. Recombinant HPP from Lactococcus lactis subsp. lactis that was expressed in Escherichia coli contained a mixture of iron and zinc in the active site and had a catalytic efficiency of ~10(3) M(-1) s(-1). Expression of the protein under iron-free conditions resulted in the production of an enzyme with a 2 order of magnitude improvement in catalytic efficiency and a mixture of zinc and manganese in the active site. Solvent isotope and viscosity effects demonstrated that proton transfer steps and product dissociation steps are not rate-limiting. X-ray structures of HPP were determined with sulfate, L-histidinol phosphate, and a complex of L-histidinol and arsenate bound in the active site. These crystal structures and the catalytic properties of variants were used to identify the structural elements required for catalysis and substrate recognition by the HPP family of enzymes within the amidohydrolase superfamily.

Structure-based activity prediction for an enzyme of unknown function.

With many genomes sequenced, a pressing challenge in biology is predicting the function of the proteins that the genes encode. When proteins are unrelated to others of known activity, bioinformatics inference for function becomes problematic. It would thus be useful to interrogate protein structures for function directly. Here, we predict the function of an enzyme of unknown activity, Tm0936 from Thermotoga maritima, by docking high-energy intermediate forms of thousands of candidate metabolites. The docking hit list was dominated by adenine analogues, which appeared to undergo C6-deamination. Four of these, including 5-methylthioadenosine and S-adenosylhomocysteine (SAH), were tested as substrates, and three had substantial catalytic rate constants (10(5) M(-1 )s(-1)). The X-ray crystal structure of the complex between Tm0936 and the product resulting from the deamination of SAH, S-inosylhomocysteine, was determined, and it corresponded closely to the predicted structure. The deaminated products can be further metabolized by T. maritima in a previously uncharacterized SAH degradation pathway. Structure-based docking with high-energy forms of potential substrates may be a useful tool to annotate enzymes for function.

Structure of Salmonella typhimurium OMP synthase in a complete substrate complex.

Dimeric Salmonella typhimurium orotate phosphoribosyltransferase (OMP synthase, EC 2.4.2.10), a key enzyme in de novo pyrimidine nucleotide synthesis, has been cocrystallized in a complete substrate E·MgPRPP·orotate complex and the structure determined to 2.2 Å resolution. This structure resembles that of Saccharomyces cerevisiae OMP synthase in showing a dramatic and asymmetric reorganization around the active site-bound ligands but shares the same basic topology previously observed in complexes of OMP synthase from S. typhimurium and Escherichia coli. The catalytic loop (residues 99-109) contributed by subunit A is reorganized to close the active site situated in subunit B and to sequester it from solvent. Furthermore, the overall structure of subunit B is more compact, because of movements of the amino-terminal hood and elements of the core domain. The catalytic loop of subunit B remains open and disordered, and subunit A retains the more relaxed conformation observed in loop-open S. typhimurium OMP synthase structures. A non-proline cis-peptide formed between Ala71 and Tyr72 is seen in both subunits. The loop-closed catalytic site of subunit B reveals that both the loop and the hood interact directly with the bound pyrophosphate group of PRPP. In contrast to dimagnesium hypoxanthine-guanine phosphoribosyltransferases, OMP synthase contains a single catalytic Mg(2+) in the closed active site. The remaining pyrophosphate charges of PRPP are neutralized by interactions with Arg99A, Lys100B, Lys103A, and His105A. The new structure confirms the importance of loop movement in catalysis by OMP synthase and identifies several additional movements that must be accomplished in each catalytic cycle. A catalytic mechanism based on enzymic and substrate-assisted stabilization of the previously documented oxocarbenium transition state structure is proposed.

Structure-based function discovery of an enzyme for the hydrolysis of phosphorylated sugar lactones.

Two enzymes of unknown function from the cog1735 subset of the amidohydrolase superfamily (AHS), LMOf2365_2620 (Lmo2620) from Listeria monocytogenes str. 4b F2365 and Bh0225 from Bacillus halodurans C-125, were cloned, expressed, and purified to homogeneity. The catalytic functions of these two enzymes were interrogated by an integrated strategy encompassing bioinformatics, computational docking to three-dimensional crystal structures, and library screening. The three-dimensional structure of Lmo2620 was determined at a resolution of 1.6 Å with two phosphates and a binuclear zinc center in the active site. The proximal phosphate bridges the binuclear metal center and is 7.1 Å from the distal phosphate. The distal phosphate hydrogen bonds with Lys-242, Lys-244, Arg-275, and Tyr-278. Enzymes within cog1735 of the AHS have previously been shown to catalyze the hydrolysis of substituted lactones. Computational docking of the high-energy intermediate form of the KEGG database to the three-dimensional structure of Lmo2620 highly enriched anionic lactones versus other candidate substrates. The active site structure and the computational docking results suggested that probable substrates would likely include phosphorylated sugar lactones. A small library of diacid sugar lactones and phosphorylated sugar lactones was synthesized and tested for substrate activity with Lmo2620 and Bh0225. Two substrates were identified for these enzymes, D-lyxono-1,4-lactone-5-phosphate and l-ribono-1,4-lactone-5-phosphate. The k(cat)/K(m) values for the cobalt-substituted enzymes with these substrates are ~10(5) M(-1) s(-1).

Conformational changes in orotidine 5'-monophosphate decarboxylase: a structure-based explanation for how the 5'-phosphate group activates the enzyme.

The binding of a ligand to orotidine 5'-monophosphate decarboxylase (OMPDC) is accompanied by a conformational change from an open, inactive conformation (E(o)) to a closed, active conformation (E(c)). As the substrate traverses the reaction coordinate to form the stabilized vinyl carbanion/carbene intermediate, interactions that destabilize the carboxylate group of the substrate and stabilize the intermediate (in the E(c)·S(‡) complex) are enforced. Focusing on the OMPDC from Methanothermobacter thermautotrophicus, we find the "remote" 5'-phosphate group of the substrate activates the enzyme 2.4 × 10(8)-fold; the activation is equivalently described by an intrinsic binding energy (IBE) of 11.4 kcal/mol. We studied residues in the activation that (1) directly contact the 5'-phosphate group, (2) participate in a hydrophobic cluster near the base of the active site loop that sequesters the bound substrate from the solvent, and (3) form hydrogen bonding interactions across the interface between the "mobile" and "fixed" half-barrel domains of the (ß/α)(8)-barrel structure. Our data support a model in which the IBE provided by the 5'-phosphate group is used to allow interactions both near the N-terminus of the active site loop and across the domain interface that stabilize both the E(c)·S and E(c)·S(‡) complexes relative to the E(o)·S complex. The conclusion that the IBE of the 5'-phosphate group provides stabilization to both the E(c)·S and E(c)·S(‡) complexes, not just the E(c)·S(‡) complex, is central to understanding the structural origins of enzymatic catalysis as well as the requirements for the de novo design of enzymes that catalyze novel reactions.

Rescue of the orphan enzyme isoguanine deaminase.

Cytosine deaminase (CDA) from Escherichia coli was shown to catalyze the deamination of isoguanine (2-oxoadenine) to xanthine. Isoguanine is an oxidation product of adenine in DNA that is mutagenic to the cell. The isoguanine deaminase activity in E. coli was partially purified by ammonium sulfate fractionation, gel filtration, and anion exchange chromatography. The active protein was identified by peptide mass fingerprint analysis as cytosine deaminase. The kinetic constants for the deamination of isoguanine at pH 7.7 are as follows: k(cat) = 49 s(-1), K(m) = 72 µM, and k(cat)/K(m) = 6.7 × 10(5) M(-1) s(-1). The kinetic constants for the deamination of cytosine are as follows: k(cat) = 45 s(-1), K(m) = 302 µM, and k(cat)/K(m) = 1.5 × 10(5) M(-1) s(-1). Under these reaction conditions, isoguanine is the better substrate for cytosine deaminase. The three-dimensional structure of CDA was determined with isoguanine in the active site.

Mechanism of the orotidine 5'-monophosphate decarboxylase-catalyzed reaction: importance of residues in the orotate binding site.

The reaction catalyzed by orotidine 5'-monophosphate decarboxylase (OMPDC) is accompanied by exceptional values for rate enhancement (k(cat)/k(non) = 7.1 × 10(16)) and catalytic proficiency [(k(cat)/K(M))/k(non) = 4.8 × 10(22) M(-1)]. Although a stabilized vinyl carbanion/carbene intermediate is located on the reaction coordinate, the structural strategies by which the reduction in the activation energy barrier is realized remain incompletely understood. This laboratory recently reported that "substrate destabilization" by Asp 70 in the OMPDC from Methanothermobacter thermoautotrophicus (MtOMPDC) lowers the activation energy barrier by ∼5 kcal/mol (contributing ~2.7 × 10(3) to the rate enhancement) [Chan, K. K., Wood, B. M., Fedorov, A. A., Fedorov, E. V., Imker, H. J., Amyes, T. L., Richard, J. P., Almo, S. C., and Gerlt, J. A. (2009) Biochemistry 48, 5518-5531]. We now report that substitutions of hydrophobic residues in a pocket proximal to the carboxylate group of the substrate (Ile 96, Leu 123, and Val 155) with neutral hydrophilic residues decrease the value of k(cat) by as much as 400-fold but have a minimal effect on the value of k(ex) for exchange of H6 of the FUMP product analogue with solvent deuterium; we hypothesize that this pocket destabilizes the substrate by preventing hydration of the substrate carboxylate group. We also report that substitutions of Ser 127 that is proximal to O4 of the orotate ring decrease the value of k(cat)/K(M), with the S127P substitution that eliminates hydrogen bonding interactions with O4 producing a 2.5 × 10(6)-fold reduction; this effect is consistent with delocalization of the negative charge of the carbanionic intermediate on O4 that produces an anionic carbene intermediate and thereby provides a structural strategy for stabilization of the intermediate. These observations provide additional information about the identities of the active site residues that contribute to the rate enhancement and, therefore, insights into the structural strategies for catalysis.

Three-dimensional structure and catalytic mechanism of cytosine deaminase.

Cytosine deaminase (CDA) from E. coli is a member of the amidohydrolase superfamily. The structure of the zinc-activated enzyme was determined in the presence of phosphonocytosine, a mimic of the tetrahedral reaction intermediate. This compound inhibits the deamination of cytosine with a K(i) of 52 nM. The zinc- and iron-containing enzymes were characterized to determine the effect of the divalent cations on activation of the hydrolytic water. Fe-CDA loses activity at low pH with a kinetic pK(a) of 6.0, and Zn-CDA has a kinetic pK(a) of 7.3. Mutation of Gln-156 decreased the catalytic activity by more than 5 orders of magnitude, supporting its role in substrate binding. Mutation of Glu-217, Asp-313, and His-246 significantly decreased catalytic activity supporting the role of these three residues in activation of the hydrolytic water molecule and facilitation of proton transfer reactions. A library of potential substrates was used to probe the structural determinants responsible for catalytic activity. CDA was able to catalyze the deamination of isocytosine and the hydrolysis of 3-oxauracil. Large inverse solvent isotope effects were obtained on k(cat) and k(cat)/K(m), consistent with the formation of a low-barrier hydrogen bond during the conversion of cytosine to uracil. A chemical mechanism for substrate deamination by CDA was proposed.