Browsing by Author "Andrews, Forest H."
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Item Identification of Charge Transfer Transitions Related to Thiamin-Bound Intermediates on Enzymes Provides a Plethora of Signatures Useful in Mechanistic Studies(American Chemical Society, 2014-04-08) Patel, Hetalben; Nemeria, Natalia S.; Andrews, Forest H.; McLeish, Michael J.; Jordan, Frank; Department of Chemistry & Chemical Biology, School of ScienceIdentification of enzyme-bound intermediates via their spectroscopic signatures, which then allows direct monitoring of the kinetic fate of these intermediates, poses a continuing challenge. As an electrophilic covalent catalyst, the thiamin diphosphate (ThDP) coenzyme forms a number of noncovalent and covalent intermediates along its reaction pathways, and multiple UV–vis and circular dichroism (CD) bands have been identified at Rutgers pertinent to several among them. These electronic transitions fall into two classes: those for which the conjugated system provides a reasonable guide to the observed λmax and others in which there is no corresponding conjugated system and the observed CD bands are best ascribed to charge transfer (CT) transitions. Herein is reported the reaction of four ThDP enzymes with alternate substrates: (a) acetyl pyruvate, its methyl ester, and fluoropyruvate, these providing the shortest side chains attached at the thiazolium C2 atom and leading to CT bands with λmax values of >390 nm, not pertinent to any on-pathway conjugated systems (estimated λmax values of <330 nm), and (b) (E)-4-(4-chlorophenyl)-2-oxo-3-butenoic acid displaying both a conjugated enamine (430 nm) and a CT transition (480 nm). We suggest that the CT transitions result from an interaction of the π bond on the ThDP C2 side chain as a donor, and the positively charged thiazolium ring as an acceptor, and correspond to covalent ThDP-bound intermediates. Time resolution of these bands allows the rate constants for individual steps to be determined. These CD methods can be applied to the entire ThDP superfamily of enzymes and should find applications with other enzymes.Item Mechanistic and Structural Insight to an Evolved Benzoylformate Decarboxylase with Enhanced Pyruvate Decarboxylase Activity(MDPI, 2016-12) Andrews, Forest H.; Wechsler, Cindy; Rogers, Megan P.; Meyer, Danilo; Tittmann, Kai; McLeish, Michael J.; Department of Chemistry and Chemical Biology, School of ScienceBenzoylformate decarboxylase (BFDC) and pyruvate decarboxylase (PDC) are thiamin diphosphate-dependent enzymes that share some structural and mechanistic similarities. Both enzymes catalyze the nonoxidative decarboxylation of 2-keto acids, yet differ considerably in their substrate specificity. In particular, the BFDC from P. putida exhibits very limited activity with pyruvate, whereas the PDCs from S. cerevisiae or from Z. mobilis show virtually no activity with benzoylformate (phenylglyoxylate). Previously, saturation mutagenesis was used to generate the BFDC T377L/A460Y variant, which exhibited a greater than 10,000-fold increase in pyruvate/benzoylformate substrate utilization ratio compared to that of wtBFDC. Much of this change could be attributed to an improvement in the Km value for pyruvate and, concomitantly, a decrease in the kcat value for benzoylformate. However, the steady-state data did not provide any details about changes in individual catalytic steps. To gain insight into the changes in conversion rates of pyruvate and benzoylformate to acetaldehyde and benzaldehyde, respectively, by the BFDC T377L/A460Y variant, reaction intermediates of both substrates were analyzed by NMR and microscopic rate constants for the elementary catalytic steps were calculated. Herein we also report the high resolution X-ray structure of the BFDC T377L/A460Y variant, which provides context for the observed changes in substrate specificity.Item Perturbation of the Monomer–Monomer Interfaces of the Benzoylformate Decarboxylase Tetramer(American Chemical Society, 2014-07-15) Andrews, Forest H.; Rogers, Megan P.; Paul, Lake N.; McLeish, Michael J.; Department of Chemistry & Chemical Biology, School of ScienceThe X-ray structure of benzoylformate decarboxylase (BFDC) from Pseudomonas putida ATCC 12633 shows it to be a tetramer. This was believed to be typical of all thiamin diphosphate-dependent decarboxylases until recently when the structure of KdcA, a branched-chain 2-keto acid decarboxylase from Lactococcus lactis, showed it to be a homodimer. This lent credence to earlier unfolding experiments on pyruvate decarboxylase from Saccharomyces cerevisiae that indicated that it might be active as a dimer. To investigate this possibility in BFDC, we sought to shift the equilibrium toward dimer formation. Point mutations were made in the noncatalytic monomer–monomer interfaces, but these had a minimal effect on both tetramer formation and catalytic activity. Subsequently, the R141E/Y288A/A306F variant was shown by analytical ultracentrifugation to be partially dimeric. It was also found to be catalytically inactive. Further experiments revealed that just two mutations, R141E and A306F, were sufficient to markedly alter the dimer–tetramer equilibrium and to provide an ∼450-fold decrease in kcat. Equilibrium denaturation studies suggested that the residual activity was possibly due to the presence of residual tetramer. The structures of the R141E and A306F variants, determined to <1.5 Å resolution, hinted that disruption of the monomer interfaces will be accompanied by movement of a loop containing Leu109 and Leu110. As these residues contribute to the hydrophobicity of the active site and the correct positioning of the substrate, it seems that tetramer formation may well be critical to the catalytic activity of BFDC.Item Phosphonodifluoropyruvate is a mechanism-based inhibitor of phosphonopyruvate decarboxylase from Bacteroides fragilis(Elsevier, 2017-08) Pallitsch, Katharina; Rogers, Megan P.; Andrews, Forest H.; Hammerschmidt, Friedrich; McLeish, Michael J.; Department of Chemistry and Chemical Biology, School of ScienceBacteroides fragilis, a human pathogen, helps in the formation of intra-abdominal abscesses and is involved in 90% of anaerobic peritoneal infections. Phosphonopyruvate decarboxylase (PnPDC), a thiamin diphosphate (ThDP)-dependent enzyme, plays a key role in the formation of 2-aminoethylphosphonate, a component of the cell wall of B. fragilis. As such PnPDC is a possible target for therapeutic intervention in this, and other phosphonate producing organisms. However, the enzyme is of more general interest as it appears to be an evolutionary forerunner to the decarboxylase family of ThDP-dependent enzymes. To date, PnPDC has proved difficult to crystallize and no X-ray structures are available. In the past we have shown that ThDP-dependent enzymes will often crystallize if the cofactor has been irreversibly inactivated. To explore this possibility, and the utility of inhibitors of phosphonate biosynthesis as potential antibiotics, we synthesized phosphonodifluoropyruvate (PnDFP) as a prospective mechanism-based inhibitor of PnPDC. Here we provide evidence that PnDFP indeed inactivates the enzyme, that the inactivation is irreversible, and is accompanied by release of fluoride ion, i.e., PnDFP bears all the hallmarks of a mechanism-based inhibitor. Unfortunately, the enzyme remains refractive to crystallization.Item Putative Benzoylformate Decarboxylases and the Annotation Problem(Office of the Vice Chancellor for Research, 2013-04-05) Malik, Ahmed M.; Logsdon, Matthew G.; Andrews, Forest H.; McLeish, Michael J.Benzoylformate decarboxylases (BFDCs) are a relatively uncommon class of thiamin diphosphate-dependent enzymes of commercial interest that catalyze the decarboxylation of benzoylformate, with BFDC from Pseudomonas putida being the most extensively studied among them. Based upon sequence homology, the recently established Thiamin Enzyme Engineering Database (TEED) has identified dozens of sequences in a variety of other microorganisms and annotated them as BFDCs. Interestingly, the majority of these putative BFDCs share >40% sequence identity with PpBFDC but many lack certain amino acids thought to be important in its function. Further, most of the annotated sequences are from organisms with no known pathway involving benzoylformate. To determine the integrity of these sequence annotations, these previously unstudied enzymes must be functionally characterized to determine if they are, in fact, true BFDCs. Currently, we are studying putative BFDCs from Polynucleobacter necessaries and Mycobacterium smegmatis, both of which share most of the same catalytic residues as PpBFDC but have alterations in residues involved in substrate specificity. We have successfully expressed, purified these supposed BFDCs, and characterized them by assaying with a variety of both metabolic and unnatural 2-ketoacid substrates. Comparison of their activity with that of PpBFDC suggests that these two sequences were both incorrectly annotated. We are currently in the process of trying to identify their natural substrate.