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Molecular characterization of NikD, a new flavoenzyme important in the biosynthesis of nikkomycin antibiotics
Journal article   Peer reviewed

Molecular characterization of NikD, a new flavoenzyme important in the biosynthesis of nikkomycin antibiotics

David Venci, Guohua Zhao and Marilyn Schuman Jorns
Biochemistry (Easton), v 41(52), pp 15795-15802
31 Dec 2002
DOI: https://doi.org/10.1021/bi020515y
PMID: 12501208
Featured in Collection :   UN Sustainable Development Goals @ Drexel

Abstract

Flavoproteins - chemistry Oxidoreductases, N-Demethylating - genetics Oxidoreductases, N-Demethylating - isolation & purification Molecular Sequence Data Substrate Specificity Aminoglycosides Paraquat - chemistry Recombinant Proteins - biosynthesis Antifungal Agents - biosynthesis Riboflavin - analogs & derivatives Recombinant Proteins - isolation & purification Flavoproteins - antagonists & inhibitors Oxidoreductases, N-Demethylating - antagonists & inhibitors Sarcosine Oxidase Light Amino Acid Sequence Recombinant Proteins - antagonists & inhibitors Riboflavin - chemistry Flavoproteins - isolation & purification Oxidation-Reduction Proline - analogs & derivatives Streptomyces - genetics Flavoproteins - genetics Proline - chemistry Oxidoreductases, N-Demethylating - chemistry Edetic Acid - chemistry Streptomyces - enzymology Anti-Bacterial Agents - biosynthesis Spectrophotometry Hydrogen-Ion Concentration Pipecolic Acids - chemistry
Nikkomycin antibiotics are potent inhibitors of chitin synthase, effective as therapeutic antifungal agents in humans and easily degradable insecticides in agriculture. NikD is a novel flavoprotein that catalyzes the oxidation of Delta(1)- or Delta(2)-piperideine-2-carboxylate, a key step in the biosynthesis of nikkomycin antibiotics. The resulting dihydropicolinate product may be further oxidized by nikD or converted to picolinate in a nonenzymic reaction. Saturated nitrogen heterocycles (L-pipecolate, L-proline) and 3,4-dehydro-L-proline act as alternate substrates. The ability of nikD to oxidize 3,4-dehydro-L-proline, but not 1-cyclohexenoate, suggests that the enzyme is specific for the oxidation of a carbon-nitrogen bond. An equivalent reaction is possible with the enamine (Delta(2)), but not the imine (Delta(1)), form of the natural piperideine-2-carboxylate substrate. Apparent steady-state kinetic parameters for the reaction of nikD with Delta(1)- or Delta(2)-piperideine-2-carboxylate (k(cat) = 64 min(-1); K(m) = 5.2 microM) or 3,4-dehydro-L-proline (k(cat) = 18 min(-1); K(m) = 13 mM) were determined in air-saturated buffer by measuring hydrogen peroxide formation in a coupled assay. NikD appears to be a new member of the monomeric sarcosine oxidase (MSOX) family of amine oxidizing enzymes. The enzyme contains 1 mol of flavin adenine dinucleotide (FAD) covalently linked to Cys321. The covalent flavin attachment site and two residues that bind substrate carboxylate in MSOX are conserved in nikD. NikD, however, exhibits an unusual long-wavelength absorption band, attributed to charge-transfer interaction between FAD and an ionizable (pK(a) = 7.3) active-site residue. Similar long-wavelength absorption bands have been observed for flavoproteins containing an active site cysteine or cysteine sulfenic acid. Interestingly, Cys273 in nikD aligns with an active-site histidine in MSOX (His269) that is, otherwise, a highly conserved residue within the MSOX family.

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Web of Science research areas
Biochemistry & Molecular Biology
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