Nikkomycin Biosynthesis:  Formation of a 4-Electron Oxidation Product during Turnover of NikD with Its Physiological Substrate

Abstract

Nikkomycins are peptidyl nucleoside antibiotics that act as therapeutic antifungal agents in humans and easily degraded insecticides in agriculture. The nikkomycin peptidyl moiety contains a pyridyl residue derived from L-lysine. The first step in peptidyl biosynthesis is an aminotransferase-catalyzed reaction that converts L-lysine to Delta(1)- or Delta(2)-piperideine-2-carboxylate (P2C). Spectral, chromatographic, and kinetic analyses show that the aerobic reaction of nikD with P2C results in the stoichiometric formation of picolinate, accompanied by the reduction of 2 mol of oxygen to hydrogen peroxide. A high resolution HPLC method, capable of separating picolinate, nicotinate and isonicotinate, was developed and used in product identification. NikD contains 1 mol of covalently bound FAD and exists as a monomer in solution. Reductive and oxidative titrations with dithionite and potassium ferricyanide, respectively, show that FAD is the only redox-active group in nikD. Anaerobic reaction of nikD with 1 mol of P2C results in immediate reduction of enzyme-bound FAD. Because nikD is an obligate 2-electron acceptor, it is proposed that the observed 4-electron oxidation of P2C to picolinate occurs via a mechanism involving two successive nikD-catalyzed 2-electron oxidation steps. In addition to nikkomycins, a nikD-like reaction is implicated in the biosynthesis of an L-lysine-derived pyridyl moiety found in streptogramin group B antibiotics that are used as part of a last resort treatment for severe infections due to gram positive bacteria.

A Mobile Tryptophan Is the Intrinsic Charge Transfer Donor in a Flavoenzyme Essential for Nikkomycin Antibiotic Biosynthesis

“…Anion exchange HPLC analyses were conducted by using isocratic elution at a flow rate of 1.4 ml/min (method 1) or 1.2 mL/min (method 2). Method 1 corresponds to the previously described high resolution protocol for separation of positional isomers of picolinate (6). The elution solvent for method 2 was prepared by mixing (v/v) 1 part of 25 mM ammonium carbonate pH 10.3 with 1 part of 50% methanol in water (v/v).…”

“…Subsequent conversion of DHP to picolinate appears to proceed without release of the labile DHP intermediate into solution (5,6).…”

3′‐Enolpyruvyl‐UMP, a Novel and Unexpected Metabolite in Nikkomycin Biosynthesis

“…Following the cloning of the entire set of structural genes involved in nikkomycin biosynthesis, [6] the enzymatic steps leading to the 4-formyl-4-imidazolin-2-one base were investigated in some detail. [7][8][9] The peptidyl moiety is synthesized by eleven enzymatic reactions, of which only two have been investigated in depth. [10,11] The aminohexuronic acid is introduced into the nikkomycin skeleton by the transfer of 5-phosphoribosyl-1-pyrophosphate to the nucleobase (uracil or 4-formyl-4-imidazolin-2-one) followed by addition of an enolpyruvyl moiety from phosphoenolpyruvate (PEP), supposedly to the 5'-hydroxyl group of the ribose.…”

Novel nikkomycin analogues generated by mutasynthesis in Streptomyces ansochromogenes

“…Previous studies demonstrated that a L-lysine 2-aminotransferase catalyzes the initial reaction from L-lysine to piperideine-2-carboxylate (P2C) in HPHT formation [9]. A monomeric sarcosine oxidases was found to be responsible for the conversion of P2C to picolinic acid, which was activated by a picolinate-CoA ligase to continue subsequent reactions [4,10]. …”