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.

Oxidative decarboxylation of pyruvate by 1-deoxy-d-xyulose 5-phosphate synthase, a central metabolic enzyme in bacteria

“…Anerobic conditions were established either in a Baker-Ruskinn in vivo (Sanford, ME) or a Coy Laboratory Products vinyl anaerobic chamber (Grass Lake, MI). E. coli WT DXP synthase and E. coli MEP synthase (IspC) were overexpressed and purified as reported previously with minor modifications for the purification of anaerobic DXP synthase (14,34). To obtain anaerobic DXP synthase, the protein was overexpressed and purified as previously described (34), however, the second dialysis was carried out in an anaerobic chamber in 50 mM HEPES, pH 8, 5% glycerol, 100 mM NaCl, 10 mM MgCl 2 , and 1 mM ThDP at 0°C for 4 h. The protein was stored in liquid N 2 until use when it was transferred directly to an anaerobic chamber.…”

Pyruvate dehydrogenase complex deficiency is linked to regulatory loop disorder in the αV138M variant of human pyruvate dehydrogenase

“…The overall activity of PDHc containing either WT or ␣V138M E1 was measured by monitoring the formation of NADH (and H ϩ ) at 340 nm, as reported previously (22). PDHc was reconstituted by premixing the E1, E2⅐E3BP, and E3 proteins at a microgram mass ratio of 1:3:3 in a buffer of 50 mM KH 2 PO 4 (pH 7.0) and 0.15 M NaCl for 60 min at 25°C (31).…”

Structural basis for antibiotic action of the B1 antivitamin 2′-methoxy-thiamine

“…1). The design principles and general modus operandi of these antivitamins are highly similar as they all bear a small chemical modification of the vitamin scaffold, are taken up by the target species in the form of a modified precursor and are eventually transformed by the native downstream machinery responsible for biosynthesis of the biologically active cofactor form, often with higher affinity than the native cofactor, to yield the mature cofactor analog [6][7][8][9][10][11] .…”

“…A set of enzyme targets for MThDP was identified in E. coli, namely α-ketoglutarate dehydrogenase, transketolase (TK) and 1-deoxy-d-xylulose-5 -phosphate synthase (DXS). In vitro studies on several ThDP enzymes demonstrated that the enzymatic activities of both E. coli pyruvate dehydrogenase (EcPDH) and E. coli 1-deoxy-d-xylulose-5 -phosphate synthase are strongly inhibited upon reconstitution with MThDP 11 . Conversely, E. coli α-ketoglutarate dehydrogenase and human PDH retain almost full enzymatic activity with MThDP as the cofactor 11 .…”

“…In vitro studies on several ThDP enzymes demonstrated that the enzymatic activities of both E. coli pyruvate dehydrogenase (EcPDH) and E. coli 1-deoxy-d-xylulose-5 -phosphate synthase are strongly inhibited upon reconstitution with MThDP 11 . Conversely, E. coli α-ketoglutarate dehydrogenase and human PDH retain almost full enzymatic activity with MThDP as the cofactor 11 . This underscores the notion that MThDP is, in principle, coenzymatically active, but it remains unclear why only some ThDP enzymes are inhibited by MThDP.…”