Phylogenetic Analysis and Functional Comparisons of Four Malate Synthase Isoforms

Malate synthase (MS), the second key enzyme in the glyoxylate cycle, catalyzes the condensation of acetyl-CoA and glyoxylate to produce malate and CoA. Phylogenetic analysis reveals four MS subgroups: MSA, MST, MSG, and MSH. Sequence alignment shows that MSA, MSG, and MSH share similar structures and active sites, while MST exhibits extremely low identity with the other three subtypes. Herein, we report the cloning, expression, and detailed characterization of MS isoforms from Acinetobacter baumannii (AbMSG), Pseudomonas aeruginosa (PaMSG), Escherichia coli (EcMSA), and Sulfolobus acidocaldarius (SaMST). AbMSG, PaMSG, and EcMSA are mesophilic, with optimum temperatures of 40°C, 32°C, and 32°C, respectively. SaMST is thermophilic, with maximal activity at 60°C, and uniquely prefers Mn²⁺, in contrast to the Mg²⁺ dependence of AbMSG, PaMSG, and EcMSA. Despite similar catalytic efficiencies for acetyl-CoA, the isoforms show pronounced differences in glyoxylate turnover. Strikingly, cis-aconitate completely inhibits MS activity, indicating this metabolite and its analogs as potential scaffolds for MS-targeted antibacterial strategies, given the pivotal role of the glyoxylate cycle in bacterial virulence.