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首页> 外文期刊>Biochemistry >Molecular characterization of NikD, a new flavoenzyme important in the biosynthesis of nikkomycin antibiotics
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Molecular characterization of NikD, a new flavoenzyme important in the biosynthesis of nikkomycin antibiotics

机译：NikD的分子表征，一种新的黄素酶，对尼克霉素抗生素的生物合成很重要

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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 (kappa_(cat) = 64 min~(-1); K_m = 5.2 muM) or 3,4-dehydro-L-proline (kappa_(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 FAS 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 (His 269) that is, otherwise, a highly conserved residue within the MSOX family.
机译：尼克霉素抗生素是几丁质合酶的有效抑制剂，可有效用作人类的治疗性抗真菌剂，并在农业中易于降解。 NikD是一种新颖的黄素蛋白，可催化DELTA〜1-或DELTA〜2-哌啶-2-羧酸的氧化，这是尼古霉素抗生素生物合成的关键步骤。所得的二氢吡啶甲酸产物可以通过nikD进一步氧化或在非酶反应中转化为吡啶甲酸。饱和氮杂环（​​L-哌酸酯，L-脯氨酸）和3,4-脱氢-L-脯氨酸可作为备用底物。 nikD氧化3,4-dehydro-L-脯氨酸而不是1-cyclohexenoate的能力表明该酶对碳氮键的氧化具有特异性。与烯胺（DELTA_2）的形式，但与亚胺（DELTA-1）的天然哌啶-2-羧酸底物形式的等效反应是可能的。 nikD与DELTA〜1-或DELTA〜2-哌啶-2-羧酸酯（kappa_（cat）= 64 min〜（-1）; K_m = 5.2μM）或3,4-的反应的表观稳态动力学参数通过在偶联试验中测量过氧化氢的形成，在空气饱和的缓冲液中测定了脱氢-L-脯氨酸（kappa_（cat）= 18 min〜（-1）; K_m = 13 mM）。 NikD似乎是胺氧化酶单体肌氨酸氧化酶（MSOX）系列的新成员。该酶含有1摩尔与Cys321共价连接的黄素腺嘌呤二核苷酸（FAD）。 nikD中保留了MSOX中的共价黄素附着位点和两个结合底物羧酸盐的残基。但是，NikD具有不寻常的长波吸收带，这归因于FAS和可电离的（pK_a = 7.3）活性位残基之间的电荷转移相互作用。对于含有活性位点半胱氨酸或半胱氨酸亚磺酸的黄素蛋白，已经观察到类似的长波吸收带。有趣的是，nikD中的Cys273与MSOX（His 269）中的一个活性位点组氨酸对齐，否则就是MSOX家族中一个高度保守的残基。

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