Saroja Narsing Rao, Mohan Anil H Shyam, Srividya D, Supreetha K
Pesticide Residue and Food Quality Analysis Laboratory, University of Agricultural Sciences, Raichur, 584104, Karnataka, India.
Department of Biotechnology, Dayananda Sagar College of Engineering, Kumaraswamy Layout, Shavige Malleswara Hills, Bengaluru, 78, Karnataka, India.
Protein Expr Purif. 2019 May;157:9-16. doi: 10.1016/j.pep.2019.01.006. Epub 2019 Jan 14.
A putrescine monooxygenase from Shewanella putrefaciens 95 (SpPMO) is the initial enzyme catalyzing the hydroxylation of putrescine to N-hydroxyl putrescine, the precursor for the synthesis of a siderophore putrebactin was identified. This PMO clustered together with known characterized NMOs from Shewanella baltica, Bordetella pertussis, Erwinia amylovora, Streptomyces sp. Gordonia rubripertincta, Pseudomonas aeruginosa and outgrouped from Escherichia coli, Nocardia farcinica, and Rhodococcus erythropolis. The deduced SpPMO protein showed 53% and 36% sequence identity with other characterized bacterial NMOs from Erwinia amylovora and Gordonia rubripertincta respectively. In this investigation, we have cloned the complete 1518bp coding sequence of pubA from Shewanella putrefaciens 95 encoding the corresponding protein SpPMO. It comprises 505 amino acid residues in length and has approximately a molecular weight of 54 kDa. Chaperone-assisted heterologous expression of SpPMO in pET151Topo expression vector under the control of bacteriophage T7 promoter permitted a stringent IPTG dependent expression. It has been successfully cloned, overexpressed and purified as a soluble His -tagged enzyme using E. coli as a cloning and expression host. The expression of recombinant SpPMO was confirmed by Western blotting using anti-His antibody. The purified protein showed FAD and NADPH dependent N-hydroxylation activity. This study has paved a way to understand the hydroxylation step of putrebactin synthesis which can be further investigated by studying its kinetic mechanism and physiological role.
从腐败希瓦氏菌95中鉴定出一种腐胺单加氧酶（SpPMO），它是催化腐胺羟基化为N-羟基腐胺的初始酶，N-羟基腐胺是铁载体腐铁菌素合成的前体。这种PMO与来自波罗的海希瓦氏菌、百日咳博德特氏菌、梨火疫欧文氏菌、链霉菌属、红橙戈登氏菌、铜绿假单胞菌的已知特征性NMO聚集在一起，并与大肠杆菌、诺卡氏菌和红平红球菌分在不同类群。推导的SpPMO蛋白与来自梨火疫欧文氏菌和红橙戈登氏菌的其他已鉴定细菌NMO分别具有53%和36%的序列同一性。在本研究中，我们克隆了来自腐败希瓦氏菌95的pubA完整1518bp编码序列，其编码相应的蛋白SpPMO。它全长505个氨基酸残基，分子量约为54 kDa。在噬菌体T7启动子控制下，SpPMO在pET151Topo表达载体中的伴侣蛋白辅助异源表达允许严格的IPTG依赖性表达。以大肠杆菌作为克隆和表达宿主，它已成功克隆、过表达并纯化成为一种可溶性的His标签酶。使用抗His抗体通过蛋白质免疫印迹法证实了重组SpPMO的表达。纯化的蛋白显示出FAD和NADPH依赖性的N-羟基化活性。这项研究为理解腐铁菌素合成的羟基化步骤铺平了道路，可通过研究其动力学机制和生理作用进一步进行研究。
