Within the DRE-TIM metallolyase superfamily, members of the Claisen-like condensation (CC-like) subgroup catalyze C–C bond-forming reactions between various α-ketoacids and acetyl-coenzyme A. These reactions are important in the metabolic pathways of many bacterial pathogens and serve as engineering scaffolds for the production of long-chain alcohol biofuels. To improve functional annotation and identify sequences that might use novel substrates in the CC-like subgroup, a combination of structural modeling and multiple-sequence alignments identified active site residues on the third, fourth, and fifth β-strands of the TIM-barrel catalytic domain that are differentially conserved within the substrate-diverse enzyme families. Using α-isopropylmalate synthase and citramalate synthase from Methanococcus jannaschii (MjIPMS and MjCMS), site-directed mutagenesis was used to test the role of each identified position in substrate selectivity. Kinetic data suggest that residues at the β3–5 and β4–7 positions play a significant role in the selection of α-ketoisovalerate over pyruvate in MjIPMS. However, complementary substitutions in MjCMS fail to alter substrate specificity, suggesting residues in these positions do not contribute to substrate selectivity in this enzyme. Analysis of the kinetic data with respect to a protein similarity network for the CC-like subgroup suggests that evolutionarily distinct forms of IPMS utilize residues at the β3–5 and β4–7 positions to affect substrate selectivity while the different versions of CMS use unique architectures. Importantly, mapping the identities of residues at the β3–5 and β4–7 positions onto the protein similarity network allows for rapid annotation of probable IPMS enzymes as well as several outlier sequences that may represent novel functions in the subgroup.

Many essential metalloproteins require iron–sulfur (Fe–S) cluster cofactors for their function. In vivo persulfide formation from l-cysteine is a key step in the biogenesis of Fe–S clusters in most organisms. In Escherichia coli, the SufS cysteine desulfurase mobilizes persulfide from l-cysteine via a PLP-dependent ping-pong reaction. SufS requires the SufE partner protein to transfer the persulfide to the SufB Fe–S cluster scaffold. Without SufE, the SufS enzyme fails to efficiently turn over and remains locked in the persulfide-bound state. Coordinated protein–protein interactions mediate sulfur transfer from SufS to SufE. Multiple studies have suggested that SufE must undergo a conformational change to extend its active site Cys loop during sulfur transfer from SufS. To test this putative model, we mutated SufE Asp74 to Arg (D74R) to increase the dynamics of the SufE Cys51 loop. Amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) analysis of SufE D74R revealed an increase in solvent accessibility and dynamics in the loop containing the active site Cys51 used to accept persulfide from SufS. Our results indicate that the mutant protein has a stronger binding affinity for SufS than that of wild-type SufE. In addition, SufE D74R can still enhance SufS desulfurase activity and did not show saturation at higher SufE D74R concentrations, unlike wild-type SufE. These results show that dynamic changes may shift SufE to a sulfur-acceptor state that interacts more strongly with SufS.

The characterization of functionally diverse enzyme superfamilies provides the opportunity to identify evolutionarily conserved catalytic strategies, as well as amino acid substitutions responsible for the evolution of new functions or specificities. Isopropylmalate synthase (IPMS) belongs to the DRE-TIM metallolyase superfamily. Members of this superfamily share common active site elements, including a conserved active site helix and an HXH divalent metal binding motif, associated with stabilization of a common enolate anion intermediate. These common elements are overlaid by variations in active site architecture resulting in the evolution of a diverse set of reactions that include condensation, lyase/aldolase, and carboxyl transfer activities. Here, using IPMS, an integrated biochemical and bioinformatics approach has been utilized to investigate the catalytic role of residues on an active site helix that is conserved across the superfamily. The construction of a sequence similarity network for the DRE-TIM metallolyase superfamily allows for the biochemical results obtained with IPMS variants to be compared across superfamily members and within other condensation-catalyzing enzymes related to IPMS. A comparison of our results with previous biochemical data indicates an active site arginine residue (R80 in IPMS) is strictly required for activity across the superfamily, suggesting that it plays a key role in catalysis, most likely through enolate stabilization. In contrast, differential results obtained from substitution of the C-terminal residue of the helix (Q84 in IPMS) suggest that this residue plays a role in reaction specificity within the superfamily.

Understanding the evolution of allostery in multidomain enzymes remains an important step in improving our ability to identify and exploit structure–function relationships in allosteric mechanisms. A recent protein similarity network for the DRE-TIM metallolyase superfamily indicated there are two evolutionarily distinct forms of the enzyme α-isopropylmalate synthase (IPMS) sharing approximately 20% sequence identity. IPMS from Mycobacterium tuberculosis has been extensively characterized with respect to catalysis and the mechanism of feedback regulation by l-leucine. Here, IPMS from Methanococcus jannaschii (MjIPMS) is used as a representative of the second form of the enzyme, and its catalytic and regulatory mechanism is compared with that of MtIPMS to identify any functional differences between the two forms. MjIPMS exhibits kinetic parameters similar to those of other reported IPMS enzymes and is partially inhibited by l-leucine in a V-type manner. Identical values of D2Okcat (3.1) were determined in the presence and absence of l-leucine, indicating the hydrolytic step is rate-determining in the absence of l-leucine and remains so in the inhibited form of the enzyme. This mechanism is identical to the mechanism identified for MtIPMS (D2Okcat = 3.3 ± 0.3 in the presence of l-leucine) despite product release being rate-determining in the uninhibited MtIPMS enzyme. The identification of identical regulatory mechanisms in enzymes with low sequence identity raises important evolutionary questions concerning the acquisition and divergence of multidomain allosteric enzymes and highlights the need for caution when comparing regulatory mechanisms for homologous enzymes.

The kinetic parameters affected by allosteric mechanisms contain collections of rate constants that vary based on differences in the relative rates of individual steps in the reaction. Thus, it may not be useful to compare enzymes with similar allosteric mechanisms unless the point of regulation has been identified. Rapid reaction kinetics and kinetic isotope effects provide a detailed description of V-type feedback allosteric inhibition in α-isopropylmalate synthase from Mycobacterium tuberculosis, an evolutionarily conserved model allosteric system. Results are consistent with a shift in the rate-determining step from product release to the hydrolytic step in catalysis in the presence of the effector.

The identification of structure–function relationships in allosteric enzymes is essential to describing a molecular mechanism for allosteric processes. The enzyme α-isopropylmalate synthase from Mycobacterium tuberculosis (MtIPMS) is subject to slow-onset, allosteric inhibition by l-leucine. Here we report that alternate amino acids act as rapid equilibrium noncompetitive inhibitors of MtIPMS failing to display biphasic inhibition kinetics. Amino acid substitutions on a flexible loop covering the regulatory binding pocket generate enzyme variants that have significant affinity for l-leucine but lack biphasic inhibition kinetics. Taken together, these results are consistent with the flexible loop mediating the slow-onset step of allosteric inhibition.

•3-Phosphoglycerate exhibits competitive substrate inhibition vs. UDP-glucose.•Solvent kinetic isotope effects suggest transfer of a proton is rate-determining.•Motifs conserved between similar GT-A enzymes can play different functional roles.Glucosyl-3-phosphoglycerate synthase (GpgS) catalyzes the first step in the biosynthesis of glucosyl glycerate, the putative precursor used in building methylated polysaccharides in mycobacteria. Enzymes from Mycobacterium tuberculosis (MtGpgS) and related species have been structurally characterized and subjected to basic kinetic analyses, but more in-depth kinetic analysis is currently lacking. Dead-end inhibition studies with MtGpgS suggest an ordered kinetic mechanism with 3-phosphoglycerate (3-PGA) binding first, followed by UDP-glucose, in contrast to previous reports. At higher concentrations, 3-PGA exhibits competitive substrate inhibition vs. UDP-glucose, suggesting 3-PGA can bind to either binding site on the enzyme. Parabolic noncompetitive inhibition plots by a 3-PGA analog also support this conclusion. The effect of varying pH on the catalytic parameters indicates single ionizable residue involved catalysis (pKa = 6.3) that must be deprotonated for full activity. A solvent kinetic isotope effect of 2.0 ± 0.3 on kcat is consistent with a proton in flight during the rate-determining step. Site-directed mutagenesis studies identify several residues critical for interactions with substrates. Although the residues are conserved among other glycosyltransferase families catalyzing similar reactions, the effect of substitutions varies between families suggesting that conserved areas play different catalytic roles in each family.