Molecular characterization of NikD, a new flavoenzyme important in the biosynthesis of nikkomycin antibiotics.
Molecular characterization of NikD, a new flavoenzyme important in the biosynthesis of nikkomycin antibiotics.
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DOI：
10.1021/bi020515y
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发表时间：
2002-12
期刊：
影响因子：
2.9
通讯作者：
David Venci;Guohua Zhao;M. Jorns
中科院分区：
文献类型：
--
作者：
David Venci;Guohua Zhao;M. Jorns
尼可霉素抗生素是几丁质合成酶的有效抑制剂，在人类中作为治疗性抗真菌剂有效，在农业中作为易降解的杀虫剂有效。NikD是一种新型黄素蛋白，其催化Delta（1）-或Delta（2）-哌啶-2-羧酸的氧化，这是尼可霉素抗生素生物合成中的关键步骤。所得的二氢吡啶甲酸酯产物可以进一步被nikD氧化或在非酶反应中转化为吡啶甲酸酯。饱和氮杂环（L-哌啶酸盐，L-脯氨酸）和3，4-脱氢-L-脯氨酸作为替代底物。nikD能够氧化3，4-脱氢-L-脯氨酸，但不能氧化1-环己烯酸，表明该酶对碳-氮键的氧化具有特异性。与天然哌啶-2-羧酸酯底物的烯胺（Delta（2））而不是亚胺（Delta（1））形式的等效反应是可能的。通过在偶联测定中测量过氧化氢形成，在空气饱和缓冲液中测定nikD与Δ（1）-或Δ（2）-哌啶-2-羧酸酯（k（cat）= 64 min（-1）; K（m）= 5.2 μ M）或3，4-脱氢-L-脯氨酸（k（cat）= 18 min（-1）; K（m）= 13 mM）反应的表观稳态动力学参数。NikD似乎是胺氧化酶单体肌氨酸氧化酶（MSOX）家族的新成员。该酶含有1摩尔的黄素腺嘌呤二核苷酸（FAD）共价连接到Cys 321。共价黄素附着位点和MSOX中结合底物羧酸的两个残基在nikD中是保守的。然而，NikD表现出不寻常的长波长吸收带，归因于FAD和可电离的（pK（a）= 7.3）活性位点残基之间的电荷转移相互作用。类似的长波长吸收带已被观察到的黄素蛋白含有一个活性位点半胱氨酸或半胱氨酸次磺酸。有趣的是，nikD中的Cys 273与MSOX中的活性位点组氨酸（His 269）比对，否则，其是MSOX家族内的高度保守残基。
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.