Division of Microbiology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, AR 72079-9502, USA.
BMC Biochem. 2010 Mar 16;11:13. doi: 10.1186/1471-2091-11-13.
Microbial degradation of azo dyes is commonly initiated by the reduction of the azo bond(s) by a group of NADH or NADPH dependant azoreductases with many requiring flavin as a cofactor. In this study, we report the identification of a novel flavin-free NADPH preferred azoreductase encoded by azoB in Pigmentiphaga kullae K24.
The deduced amino acid sequence of azoB from P. kullae K24 showed 61% identity to a previously studied azoreductase (AzoA) from the same strain. azoB encoded a protein of 203 amino acids and heterologously expressed in Escherichia coli. The purified recombinant enzyme was a monomer with a molecular mass of 22 kDa. Both NADH and NADPH can be used as an electron donor for its activity with 4-(4-hydroxy-1-naphthylazo) benzenesulfonic acid (Orange I) as substrate. The apparent Km values for both NADH and Orange I were 170 and 8.6 microM, respectively. The Km of NADPH for the enzyme is 1.0 microM. When NADPH served as the electron donor, the activity of the enzyme is 63% higher than that when NADH was used. The pH and temperature optima for activity of the enzyme with Orange I as the substrate were at pH 6.0 and between 37 and 45 degrees C. Phylogenetic analysis shows that AzoB belongs to the flavin-free azoreductase group which has a key fingerprint motif GXXGXXG for NAD(P)H binding at the N-terminus of the amino acid sequences. The 3D structure of AzoB was generated by comparative modeling approach. The structural combination of three conserved glycine residues (G7xxG10xxG13) in the pyrophosphate-binding loop with the Arg-32 explains the preference for NADPH of AzoB.
The biochemical and structural properties of AzoB from P. kullae K24 revealed its preference for NADPH over NADH and it is a member of the monomeric flavin-free azoreductase group. Our studies show the substrate specificity of AzoB based on structure and cofactor requirement and the phylogenetic relationship among azoreductase groups.
微生物对偶氮染料的降解通常是由一组依赖 NADH 或 NADPH 的偶氮还原酶启动的，其中许多还原酶需要黄素作为辅因子。在本研究中，我们报道了在 Pigmentiphaga kullae K24 中发现的一种新型黄素非依赖型 NADPH 偏好偶氮还原酶，其由 azoB 编码。
从 Pigmentiphaga kullae K24 中推测出的 azoB 氨基酸序列与同一菌株中先前研究的偶氮还原酶（AzoA）具有 61%的同一性。azoB 编码一个由 203 个氨基酸组成的蛋白质，并在大肠杆菌中异源表达。纯化的重组酶是一个单体，分子量为 22kDa。NADH 和 NADPH 都可以作为其活性的电子供体，以 4-（4-羟基-1-萘基偶氮）苯磺酸（Orange I）为底物。该酶对 NADH 和 Orange I 的表观 Km 值分别为 170 和 8.6 μM。该酶对 NADPH 的 Km 值为 1.0 μM。当 NADPH 作为电子供体时，酶的活性比使用 NADH 时高 63%。以 Orange I 为底物时，酶活性的 pH 和温度最佳值分别为 pH 6.0 和 37-45°C。系统发育分析表明，AzoB 属于黄素非依赖型偶氮还原酶组，其在氨基酸序列的 N 端具有关键的指纹基序 GXXGXXG，用于 NAD（P）H 结合。通过比较建模方法生成了 AzoB 的 3D 结构。焦磷酸结合环中三个保守甘氨酸残基（G7xxG10xxG13）与 Arg-32 的结构组合解释了 AzoB 对 NADPH 的偏好。
来自 Pigmentiphaga kullae K24 的 AzoB 的生化和结构特性表明其对 NADPH 的偏好高于 NADH，并且它是单体黄素非依赖型偶氮还原酶组的成员。我们的研究根据结构和辅因子要求以及偶氮还原酶组之间的系统发育关系显示了 AzoB 的底物特异性。
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