title: Competence of Thiamin Diphosphate-Dependent Enzymes with 2'-Methoxythiamin Diphosphate Derived from Bacimethrin, a Naturally Occurring Thiamin Anti-vitamin.
abstract: Bacimethrin (4-amino-5-hydroxymethyl-2-methoxypyrimidine), a natural product isolated from some bacteria, has been implicated as an inhibitor of bacterial and yeast growth, as well as in inhibition of thiamin biosynthesis. Given that thiamin biosynthetic enzymes could convert bacimethrin to 2'-methoxythiamin diphosphate (MeOThDP), it is important to evaluate the effect of this coenzyme analogue on thiamin diphosphate (ThDP)-dependent enzymes. The potential functions of MeOThDP were explored on five ThDP-dependent enzymes: the human and Escherichia coli pyruvate dehydrogenase complexes (PDHc-h and PDHc-ec, respectively), the E. coli 1-deoxy-D-xylulose 5-phosphate synthase (DXPS), and the human and E. coli 2-oxoglutarate dehydrogenase complexes (OGDHc-h and OGDHc-ec, respectively). Using several mechanistic tools (fluorescence, circular dichroism, kinetics, and mass spectrometry), it was demonstrated that MeOThDP binds in the active centers of ThDP-dependent enzymes, however, with a binding mode different from that of ThDP. While modest activities resulted from addition of MeOThDP to E. coli PDHc (6-11%) and DXPS (9-14%), suggesting that MeOThDP-derived covalent intermediates are converted to the corresponding products (albeit with rates slower than that with ThDP), remarkably strong activity (up to 75%) resulted upon addition of the coenzyme analogue to PDHc-h. With PDHc-ec and PDHc-h, the coenzyme analogue could support all reactions, including communication between components in the complex. No functional substitution of MeOThDP for ThDP was in evidence with either OGDH-h or OGDH-ec, shown to be due to tight binding of ThDP.
authors.affiliation: Department of Chemistry, Rutgers University , Newark, New Jersey 07102, United States.
authors.forename: Natalia S
authors.initials: NS
authors.lastname: Nemeria
authors.affiliation: Department of Chemistry, Texas A&M University , College Station, Texas 77842, United States.
authors.forename: Brateen
authors.initials: B
authors.lastname: Shome
authors.affiliation: Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine , Baltimore, Maryland 21205, United States.
authors.forename: Alicia A
authors.initials: AA
authors.lastname: DeColli
authors.affiliation: Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine , Baltimore, Maryland 21205, United States.
authors.forename: Kathryn
authors.initials: K
authors.lastname: Heflin
authors.affiliation: Department of Chemistry, Texas A&M University , College Station, Texas 77842, United States.
authors.forename: Tadhg P
authors.initials: TP
authors.lastname: Begley
authors.affiliation: Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine , Baltimore, Maryland 21205, United States.
authors.forename: Caren Freel
authors.initials: CF
authors.lastname: Meyers
authors.affiliation: Department of Chemistry, Rutgers University , Newark, New Jersey 07102, United States.
authors.forename: Frank
authors.initials: F
authors.lastname: Jordan
mesh_terms: D019943:Amino Acid Substitution; D001051:Apoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001667:Binding, Competitive; D055162:Biocatalysis; D020134:Catalytic Domain; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007655:Ketoglutarate Dehydrogenase Complex / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011743:Pyrimidines / Q000737:chemistry; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013835:Thiamine Pyrophosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D014166:Transferases / Q000737:chemistry / Q000378:metabolism*
chemical_list: C442364:2'-methoxythiamine pyrophosphate; D001051:Apoenzymes; D029968:Escherichia coli Proteins; D021122:Protein Subunits; D011743:Pyrimidines; D011768:Pyruvate Dehydrogenase Complex; D011994:Recombinant Proteins; C054356:bacimethrin; D007655:Ketoglutarate Dehydrogenase Complex; C553252:sucB protein, E coli; D014166:Transferases; C109461:deoxyxylulose-5-phosphate synthase; D013835:Thiamine Pyrophosphate
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