Prasad U Venkateswara, Vasu D, Gowtham R Rishi, Pradeep Ch Krishna, Swarupa V, Yeswanth S, Choudhary Abhijit, Sarma P V G K
Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India.
Department of Microbiology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India.
Adv Biomed Res. 2017 Jul 31;6:97. doi: 10.4103/2277-9175.211833. eCollection 2017.
has the ability to form biofilms on any niches, a key pathogenic factor of this organism and this phenomenon is directly related to the concentration of NADPH. The formation of NADP is catalyzed by NAD kinase (NADK) and this gene of ATCC 12600 was cloned, sequenced, expressed and characterized.
The NADK gene was polymerase chain reaction amplified from the chromosomal DNA of ATCC 12600 and cloned in pQE 30 vector, sequenced and expressed in DH5α. The pure protein was obtained by passing through nickel metal chelate agarose column. The enzyme kinetics of the enzyme and biofilm assay of the was carried out in both aerobic and anaerobic conditions. The kinetics was further confirmed by the ability of the substrates to dock to the NADK structure.
The recombinant NADK exhibited single band with a molecular weight of 31kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gene sequence (GenBank: JN645814) revealed presence of only one kind of NADK in all strains. The enzyme exhibited very high affinity for NAD compared to adenosine triphosphate concurring with the docking results. A root-mean-square deviation value 14.039Å observed when NADK structure was superimposed with its human counterpart suggesting very low homology. In anaerobic conditions, higher biofilm units were found with decreased NADK activity.
The results of this study suggest increased NADPH concentration in plays a vital role in the biofilm formation and survival of this pathogen in any environmental conditions.
[该生物]有能力在任何生态位形成生物膜，这是该生物体的一个关键致病因素，且这种现象与烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的浓度直接相关。烟酰胺腺嘌呤二核苷酸磷酸（NADP）的形成由烟酰胺腺嘌呤二核苷酸激酶（NADK）催化，已对ATCC 12600的该基因进行了克隆、测序、表达及特性鉴定。
从ATCC 12600的染色体DNA中通过聚合酶链反应扩增NADK基因，并克隆到pQE 30载体中，在DH5α中进行测序和表达。通过镍金属螯合琼脂糖柱获得纯蛋白。在有氧和厌氧条件下均对该酶进行酶动力学及生物膜检测。通过底物与NADK结构对接的能力进一步证实动力学情况。
重组NADK在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳中呈现分子量为31kDa的单一条带，基因序列（GenBank：JN645814）显示所有菌株中仅存在一种NADK。与三磷酸腺苷相比，该酶对烟酰胺腺嘌呤二核苷酸（NAD）表现出非常高的亲和力，这与对接结果一致。当NADK结构与其人类对应物叠加时，观察到均方根偏差值为14.039Å，表明同源性非常低。在厌氧条件下，发现生物膜单位增加而NADK活性降低。
本研究结果表明，[该生物]中烟酰胺腺嘌呤二核苷酸磷酸（NADPH）浓度的增加在该病原体在任何环境条件下的生物膜形成和存活中起着至关重要的作用。
