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Provost, J. J.

Publications and source records attributed to Provost, J. J..

2 recordsLinked to original sources
bioRxiv2025

Molecular Architecture of the human Citrate Synthase-Malate Dehydrogenase 2 metabolon

Metabolons are transient biomolecular complexes that enhance the efficiency of metabolic pathways through substrate channeling. These complexes are difficult to study because of the transient nature, thus limiting our understanding of how they are formed and regulated. The citric acid cycle is proposed to contain many such complexes although few have been characterized structurally. Here, we provide direct structural evidence for the complex of human Citrate Synthase and human mitochondrial Malate Dehydrogenase 2, which is part of the larger proposed citric acid cycle metabolon. Our structural model supports previous crosslinking studies and suggests that hMDH2 can interact with each subunit of the hCS dimer, forming up to a hexameric complex. However, this complex appears transient as titration of hMDH2 into hCS in activity assays does not saturate. We further show that the interaction site with hCS is non-specific, as hCS could also stimulate oxaloacetate formation by cytosolic and plant MDH enzymes. This structural model will provide a base for understanding the structure and regulation of the broader citric acid cycle metabolon.

biochemistry↗
bioRxiv2025

Structural and enzymatic divergence between human MDH isoforms underlies their specialized regulatory roles in metabolism

Malate dehydrogenase (MDH: EC:1.1.1.37) catalyzes a key NAD+-dependent redox reaction integral to cellular metabolism. In humans, the cytosolic (hMDH1) and mitochondrial (hMDH2) isoforms operate in distinct compartments, suggesting potential differences in regulation. Here, we present a comparative analysis of hMDH1 and hMDH2 under physiologically relevant conditions, integrating enzymatic assays, ligand binding studies, small-angle X-ray scattering (SAXS), and molecular modeling. Our findings reveal that hMDH2 activity is inhibited by -ketoglutarate, glutamate, NAD+, ATP, and citrate at concentrations consistent with mitochondrial metabolic states characterized by elevated amino acid catabolism or redox stress. Conversely, hMDH1 exhibits minimal impact by these metabolites, with only modest inhibition observed in the presence of ATP and ADP. SAXS analyses confirm that both isoforms maintain stable dimeric structures upon ligand binding, indicating that regulation is not mediated by global conformational changes. Structural modeling and normal mode analyses identify increased flexibility in hMDH1, particularly within the active site loop, thumb loop, and a partially disordered C-terminal helix. In contrast, hMDH2 displays a more rigid architecture and a more electropositive active site environment, correlating with its heightened sensitivity to anionic metabolites. Fluorescence quenching experiments further support these distinctions, demonstrating stronger binding affinities for nucleotide-based ligands in hMDH2 compared to hMDH1. Collectively, these results suggest that isoform-specific regulation of human MDH arises from differences in local structural dynamics and electrostatics, rather than large-scale structural rearrangements. hMDH2 appears adapted to integrate mitochondrial metabolic signals, modulating malate oxidation in response to cellular conditions, while hMDH1 maintains consistent cytosolic function across diverse metabolic states.

biochemistry↗