菌株Alcaligenes sp.P156中一个新型烟酸羟化酶的克隆与功能验证

本研究预测naa cluster中的naaB基因负责催化烟酸到6-羟基烟酸的反应。通过对NaaB的蛋白序列进行分析,发现该酶含有三个亚基,分别由naaB_L1、naaB_S、naaB_L2基因编码,其中naaB_L1和naaB_L2分别编码2个含有钼辅因子结合结构的大亚基,naaB_S基因编码一个含有2Fe-2S]簇的小亚基。本研究对naaB基因进行了克隆,将构建的重组质粒pME6032-naaB_L1SL2分别转化到无色杆菌(Achromobacter sp.)和大肠杆菌(Escherichia coli)中,通过HPLC和LC-MS检测验证了NaaB负责催化烟酸到6-羟基烟酸,并且在无色杆菌和大肠杆菌中NaaB均能够转化烟酸,这与之前报道烟酸羟化酶不能在大肠杆菌表达的结果不同,说明NaaB的基因编码和催化功能具有独特性。通过序列分析推测NaaB的这一特性可能跟该酶同时含有两个钼结合结构域的大亚基有关。

Cloning and Characterization of a Three Subunits Molybdenum-Containing Nicotinic Acid Hydroxylase from Alcaligenes sp .P156

Alcaligenes sp.P156 can utilize nicotinamide as its sole source of carbon,nitrogen and energy.A gene cluster naa cluster related to nicotinamide degradation has been identified,and the molecular mechanism of nicotinamide degradation has been studied.However,the molecular mechanism of nicotinic acid transformation was unclear.This study predicted that naaB gene in nna cluster was responsible for the conversion from nicotinic acid to 6-hydroxynicotinic acid.The protein sequence analysis of NaaB showed that the enzyme contained three subunits,which were encoded by naaB_L1,naaB_S and naaB_L2 genes respectively.The naaB_L1 and naaB_L2 genes encoded two large subunits containing molybdenum cofactor binding structure,respectively.The naaB_S gene encoded a small subunit containing2Fe-2S cluster.The nicotinic acid hydroxylase gene naaB was amplified,and the recombinant plasmid pME6032-naaB_L1SL2 was transformed into Achromobacter sp.SJY1 and Escherichia coli Trans1-T1 respectively to verify the transformation of nicotinic acid.The results of HPLC and LC-MS showed that NaaB was responsible for catalyzing nicotinic acid to 6-hydroxynicotinic acid,and NaaB in Achromobacter sp.and Escherichia coli could transform nicotinic acid,which was different from the previous report that nicotinic acid hydroxylase could not be expressed in Escherichia coli.This result indicated that the gene coding and catalytic function of NaaB are unique.Sequence analysis suggested that this characteristic of NaaB may be related to the fact that it contains two large subunits of molybdenum binding domain.Therefore,this study contributes to the knowledge on the degradation of pyridinic compounds.