Biotin-dependent multifunctional enzymes carry out metabolically important carboxyl group transfer reactions and are potential targets for the treatment of obesity and type 2 diabetes. These enzymes use a tethered biotin cofactor to carry an activated carboxyl group between distantly spaced active sites. The mechanism of this transfer has remained poorly understood. Here we report the complete structure of pyruvate carboxylase at 2.0 angstroms resolution, which shows its domain arrangement. The structure, when combined with mutagenic analysis, shows that intermediate transfer occurs between active sites on separate polypeptide chains. In addition, domain rearrangements associated with activator binding decrease the distance between active-site pairs, providing a mechanism for allosteric activation. This description provides insight into the function of biotin-dependent enzymes and presents a new paradigm for multifunctional enzyme catalysis.
Oxalate decarboxylase (OxDC) catalyzes a remarkable transformation in which the C-C bond in oxalate is cleaved to give carbon dioxide and formate. Like the native OxDC isolated from Aspergillus niger, the recombinant, bacterial OxDC from Bacillus subtilis contains Mn(II) in its resting state and requires catalytic dioxygen for activity. The most likely mechanism for OxDC-catalyzed C-C bond cleavage involves the participation of free radical intermediates, although this hypothesis remains to be unequivocally demonstrated. Efforts to delineate the catalytic mechanism have been placed on a firm foundation by the high-resolution crystal structure of recombinant, wild type B. subtilis OxDC (Anand et al., Biochemistry 2002, 41, 7659-7669). We now report the results of heavy-atom kinetic isotope effect measurements for the OxDC-catalyzed decarboxylation of oxalate, in what appear to be the first detailed studies of the mechanism employed by OxDC. At pH 4.2, the OxDC-catalyzed formation of formate and CO(2) have normal (13)C isotope effects of 1.5% +/- 0.1% and 0.5% +/- 0.1%, respectively, while the (18)O isotope effect on the formation of formate is 1.1% +/- 0.2% normal. Similarly at pH 5.7, the production of formate and CO(2) exhibits normal (13)C isotope effects of 1.9% +/- 0.1% and 0.8% +/- 0.1%, respectively, and the (18)O isotope effect on the formation of formate is 1.0% +/- 0.2% normal. The (18)O isotope effect on the formation of CO(2), however, 0.7% +/- 0.2%, is inverse at pH 5.7. These results are consistent with a multistep model in which a reversible, proton-coupled, electron transfer from bound oxalate to the Mn-enzyme gives an oxalate radical, which decarboxylates to yield a formate radical anion. Subsequent reduction and protonation of this intermediate then gives formate.
Oxalate decarboxylase (OxDC) catalyzes the conversion of oxalate into CO(2) and formate using a catalytic mechanism that remains poorly understood. The Bacillus subtilis enzyme is composed of two cupin domains, each of which contains Mn(II) coordinated by four conserved residues. We have measured heavy atom isotope effects for a series of Bacillus subtilis OxDC mutants in which Arg-92, Arg-270, Glu-162, and Glu-333 are conservatively substituted in an effort to define the functional roles of these residues. This strategy has the advantage that observed isotope effects report directly on OxDC molecules in which the active site manganese center(s) is (are) catalytically active. Our results support the proposal that the N-terminal Mn-binding site can mediate catalysis, and confirm the importance of Arg-92 in catalytic activity. On the other hand, substitution of Arg-270 and Glu-333 affects both Mn(II) incorporation and the ability of Mn to bind to the OxDC mutants, thereby precluding any definitive assessment of whether the metal center in the C-terminal domain can also mediate catalysis. New evidence for the importance of Glu-162 in controlling metal reactivity has been provided by the unexpected observation that the E162Q OxDC mutant exhibits a significantly increased oxalate oxidase and a concomitant reduction in decarboxylase activities relative to wild type OxDC. Hence the reaction specificity of a catalytically active Mn center in OxDC can be perturbed by relatively small changes in local protein environment, in agreement with a proposal based on prior computational studies.
Orotidine 5'-monophosphate decarboxylase has been heavily examined in recent years due to its enzymatic proficiency, which provides a catalytic enhancement to a reaction rate approximately 1017 times greater than that of the nonenzymatic reaction. Several mechanisms proposed to explain this catalytic enhancement have included covalent addition, ylide or carbene formation, and most recently concerted protonation. All of these mechanisms have circumvented the formation of a high-energy vinyl anionic intermediate. To investigate the presence of an anionic intermediate, 13C isotope effect studies have been performed using the alternate substrate 5-fluoro-OMP (OMP = orotidine 5'-monophosphate). Isotope effects obtained for the wild-type enzyme with OMP and 5-fluoro-OMP are 1.0255 and 1.0106, respectively, corresponding to a decrease of approximately 1.5% for 5-fluoro-OMP. With the K59A enzyme, the intrinisic isotope effects show a similar decrease of approximately 1.9% from 1.0543 with OMP to 1.0356 with 5-fluoro-OMP. This decrease results from the inductive effect of the fluorine, which stabilizes the carbanion intermediate by electron withdrawal and produces a reaction with an earlier transition state. The isotope effect for the decarboxylation of the slow substrate 2'-deoxy-OMP produced a intrinsic isotope effect of nearly 1.0461.
15N isotope effects and solvent deuterium isotope effects have been measured for the hydrolytic deamination of cytidine catalyzed by Escherichia coli cytidine deaminase and for the uncatalyzed reaction proceeding spontaneously in neutral solution at elevated temperatures. The primary (15)(V/K) arising from the exocyclic amino group for wild-type cytidine deaminase acting on its natural substrate, cytidine, is 1.0109 (in H(2)O, pH 7.3), 1.0123 (in H(2)O, pH 4.2), and 1.0086 (in D(2)O, pD 7.3). Increasing solvent D(2)O content has no substantial effect on k(cat) but enhances k(cat)/K(m), with a proton inventory showing that the fractionation factors of at least two protons increase markedly during the reaction. Mutant cytidine deaminases with reduced catalytic activity show more pronounced (15)N isotope effects of 1.0124 (Glu91Ala), 1.0134 (His102Ala), and 1.0158 (His102Asn) at pH 7.3 in H(2)O, as expected for processes in which the chemical transformation of the substrate becomes more rate determining. The isotope effect of mutant His102Asn is 1.033 after correcting for protonation of the -NH(2) group, and represents the intrinsic isotope effect on C-N bond cleavage. This result allows an estimation of the forward commitment of the reaction with the wild-type enzyme. The observed (15)N kinetic isotope effect of the pyrimidine N-3, for wild-type cytidine deaminase acting on cytidine, is 0.9879, which is consistent with protonation and rehybidization of N-3 with hydroxide ion attack on the adjacent carbon to create a tetrahedral intermediate. These results show that enzymatic deamination of cytidine proceeds stepwise through a tetrahedral intermediate with ammonia elimination as the major rate-determining step. The primary (15)N isotope effects observed for the uncatalyzed reaction at pH 7 (1.0021) and pH 12.5 (1.0034) were found to be insensitive to changing temperatures between 100 and 185 degrees C. These results show that the uncatalyzed and the enzymatic deaminations of cytidine proceed by similar mechanisms, although the commitment to C-N bond breaking is greater for the spontaneous reaction.
The conformational properties of an active-site loop segment, defined by residues Ser161-Glu162-Asn163-Ser164, have been shown to be important for modulating the intrinsic reactivity of Mn(II) in the active site of Bacillus subtilis oxalate decarboxylase. We now detail the functional and structural consequences of removing a conserved Arg/Thr hydrogen bonding interaction by site-specific mutagenesis. Hence, substitution of Thr-165 by a valine residue gives an OxDC variant (T165V) that exhibits impaired catalytic activity. Heavy-atom isotope effect measurements, in combination with the X-ray crystal structure of the T165V OxDC variant, demonstrate that the conserved Arg/Thr hydrogen bond is important for correctly locating the side chain of Glu-162, which mediates a proton-coupled electron transfer (PCET) step prior to decarboxylation in the catalytically competent form of OxDC. In addition, we show that the T165V OxDC variant exhibits a lower level of oxalate consumption per dioxygen molecule, consistent with the predictions of recent spin-trapping experiments (Imaram et al., 2011, Free Rad. Biol. Med. 50, 1009–1015). This finding implies that dioxygen might participate as a reversible electron sink in two putative PCET steps and is not merely used to generate a protein-based radical or oxidized metal center.
N-Acetylperosamine is an unusual dideoxysugar found in the O-antigens of some Gram-negative bacteria, including the pathogenic Escherichia coli strain O157:H7. The last step in its biosynthesis is catalyzed by PerB, an N-acetyltransferase belonging to the left-handed β-helix superfamily of proteins. Here we describe a combined structural and functional investigation of PerB from Caulobacter crescentus. For this study, three structures were determined to 1.0 Å resolution or better: the enzyme in complex with CoA and GDP-perosamine, the protein with bound CoA and GDP-N-acetylperosamine, and the enzyme containing a tetrahedral transition state mimic bound in the active site. Each subunit of the trimeric enzyme folds into two distinct regions. The N-terminal domain is globular and dominated by a six-stranded mainly parallel β-sheet. It provides most of the interactions between the protein and GDP-perosamine. The C-terminal domain consists of a left-handed β-helix, which has nearly seven turns. This region provides the scaffold for CoA binding. On the basis of these high-resolution structures, site-directed mutant proteins were constructed to test the roles of His 141 and Asp 142 in the catalytic mechanism. Kinetic data and pH−rate profiles are indicative of His 141 serving as a general base. In addition, the backbone amide group of Gly 159 provides an oxyanion hole for stabilization of the tetrahedral transition state. The pH−rate profiles are also consistent with the GDP-linked amino sugar substrate entering the active site in its unprotonated form. Finally, for this investigation, we show that PerB can accept GDP-3-deoxyperosamine as an alternative substrate, thus representing the production of a novel trideoxysugar.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.