title: Influence of allosteric regulators on individual steps in the reaction catalyzed by Mycobacterium tuberculosis 2-hydroxy-3-oxoadipate synthase.
abstract: Allosteric regulation often controls key branch points in metabolic processes. Mycobacterium tuberculosis 2-hydroxy-3-oxoadipate synthase (HOAS), a thiamin diphosphate (ThDP)-dependent enzyme, produces 2-hydroxy-3-oxoadipate using 2-ketoglutarate and glyoxylate. The proposed chemical mechanism in analogy with other ThDP-dependent carboligases involves multiple ThDP-bound covalent intermediates. Acetyl coenzyme A is an activator, and GarA, a forkhead association domain-containing protein known to regulate glutamate metabolism, is an allosteric inhibitor of HOAS. Steady state kinetics using assays to study the first half and the full catalytic cycle suggested that the regulators act at different steps in the overall mechanism. To explore the modes of regulation and to test the effects on individual catalytic steps, we performed circular dichroism (CD) studies using a non-decarboxylatable 2-ketoglutarate analog and determined the distribution of ThDP-bound covalent intermediates during the steady state of the HOAS reaction using one-dimensional (1)H gradient carbon heteronuclear single quantum coherence NMR. The results suggest that acetyl coenzyme A acts as a mixed V and K type activator and predominantly affects the predecarboxylation steps. GarA does not inhibit the formation of the predecarboxylation analog and does not affect the accumulation of the postdecarboxylation covalent intermediate derived from 2-ketoglutarate; however, it decreases the abundance of the product ThDP adduct in the HOAS pathway. Thus, the two regulators act on different halves of the catalytic cycle in an unusual regulatory regime.
authors.affiliation: Department of Microbiology and Immunology, Weill Cornell Medical College, New York, New York 10065, USA.
authors.forename: Anand
authors.initials: A
authors.lastname: Balakrishnan
authors.forename: Frank
authors.initials: F
authors.lastname: Jordan
authors.forename: Carl F
authors.initials: CF
authors.lastname: Nathan
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism; D000272:Adipates / Q000737:chemistry / Q000378:metabolism; D019880:Aldehyde-Ketone Transferases; D000465:Algorithms; D000494:Allosteric Regulation; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000378:metabolism; D006038:Glyoxylates / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007656:Ketoglutaric Acids / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D015394:Molecular Structure; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000378:metabolism*; D007652:Oxo-Acid-Lyases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013835:Thiamine Pyrophosphate / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000272:Adipates; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D006038:Glyoxylates; D007656:Ketoglutaric Acids; C027316:3-oxoadipic acid; D000105:Acetyl Coenzyme A; D019880:Aldehyde-Ketone Transferases; C021414:2-hydroxy-3-oxoadipate synthase; D007652:Oxo-Acid-Lyases; C031150:glyoxylic acid; D013835:Thiamine Pyrophosphate
keywords: 2-Hydroxy-3-oxoadipate Synthase; Acetyl Coenzyme A; Allosteric Regulation; Bacteria; Metabolism; Mycobacterium tuberculosis; Thiamin Diphosphate-dependent Enzymes