Abstract
Bacterial hybrid malic enzymes (MaeB grouping, multidomain) catalyse the transformation of malate to pyruvate, and are a major contributor to cellular reducing power and carbon flux. Distinct from other malic enzyme subtypes, the hybrid enzymes are regulated by acetyl-CoA, a molecular indicator of the metabolic state of the cell. Here we solve the structure of a MaeB protein, which reveals hybrid enzymes use the appended phosphotransacetylase (PTA) domain to form a hexameric sensor that communicates acetyl-CoA occupancy to the malic enzyme active site, 60 Å away. We demonstrate that allostery is governed by a large-scale rearrangement that rotates the catalytic subunits 70° between the two states, identifying MaeB as a new model enzyme for the study of ligand-induced conformational change. Our work provides the mechanistic basis for metabolic control of hybrid malic enzymes, and identifies inhibition-insensitive variants that may find utility in synthetic biology.
Highlights
Bacterial hybrid malic enzymes (MaeB grouping, multidomain) catalyse the transformation of malate to pyruvate, and are a major contributor to cellular reducing power and carbon flux
From structures of both bound and active states of the isolated domains and full-length enzyme, we reveal that acetyl-CoA, a potent allosteric inhibitor of Malic enzymes (MEs) activity, interacts with a conserved pocket of the PTA domain, driving conformational changes that are propagated 60 Å to the ME active site
MaeBME closely resembles the fold of small prokaryotic MEs (Fig. 1B), which form a tightly interacting dimer where the catalytic site is composed of residues from both protomers[26]
Summary
Bacterial hybrid malic enzymes (MaeB grouping, multidomain) catalyse the transformation of malate to pyruvate, and are a major contributor to cellular reducing power and carbon flux. Distinct from other malic enzyme subtypes, the hybrid enzymes are regulated by acetyl-CoA, a molecular indicator of the metabolic state of the cell. We solve the structure of a MaeB protein, which reveals hybrid enzymes use the appended phosphotransacetylase (PTA) domain to form a hexameric sensor that communicates acetyl-CoA occupancy to the malic enzyme active site, 60 Å away. The homologous TME is suggested to function instead as an NADPH and acetylCoA generator (required for anabolic reactions, the latter made indirectly), similar to other NADP+ specific enzymes like E. coli MaeB23. These products provide a carbon flux to phosphoenolpyruvate, for gluconeogenesis. A C-terminal truncation of MaeB was unable to oligomerise and became insensitive to acetyl-CoA inhibition, suggesting that allosteric regulation is mediated by the PTA domain[23]
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