Lakshmi Hanumanthu Prasanna, Yeswanth Sthanikam, Prasad Uppu Venkateswara, Vasu Dudipeta, Swarupa Vimjam, Kumar Pasupuleti Santhosh, Narasu Mangamoori Lakshmi, Krishna Sarma Potukuchi Venkata Gurunadha
Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati, AP, India 517 507.
Bioinformation. 2013;9(4):169-73. doi: 10.6026/97320630009169. Epub 2013 Feb 21.
Glucose-6-phosphate (G-6-P) formation in Staphylococcus aureus is catalysed by glucokinase (glkA) gene under high glucose concentration leading to upregulation of various pathogenic factors; therefore the present study is aimed in the cloning and characterization of glk A gene from S. aureus ATCC12600. The glk A gene was cloned in the Sma I site of pQE 30, sequenced (Accession number: JN645812) and expressed in E. coli DH5α. The recombinant glk A expressed from the resultant glk A 1 clone was purified using nickel metal chelate chromatography, the pure enzyme gave single band in SDS-PAGE with molecular weight of 33kDa. The rglk A showed very high affinity to glucose Km 5.1±0.06mM with Hill coefficient of 1.66±0.032mM. Analysis of glucokinase sequence of S. aureus showed presence of typical ATP binding site and ROK motif CNCGRSGCIE. Sequentially and phylogenetically S. aureus glk A exhibited low identity with other bacterial glk A and 21% homology with human glucokinase (GCK). Functionally, S. aureus glk A showed higher rate of G-6-P formation compared to human GCK which may have profound role in the pathogenesis.
在高葡萄糖浓度下，金黄色葡萄球菌中的葡萄糖-6-磷酸（G-6-P）形成由葡萄糖激酶（glkA）基因催化，导致各种致病因子上调；因此，本研究旨在克隆和鉴定来自金黄色葡萄球菌ATCC12600的glkA基因。将glkA基因克隆到pQE 30的Sma I位点，进行测序（登录号：JN645812）并在大肠杆菌DH5α中表达。使用镍金属螯合色谱法纯化从所得glkA 1克隆表达的重组glkA，纯酶在SDS-PAGE中呈现单一条带，分子量为33kDa。rglkA对葡萄糖表现出非常高的亲和力，Km为5.1±0.06mM，希尔系数为1.66±0.032mM。对金黄色葡萄球菌葡萄糖激酶序列的分析表明存在典型的ATP结合位点和ROK基序CNCGRSGCIE。在序列和系统发育上，金黄色葡萄球菌glkA与其他细菌的glkA具有较低的同一性，与人类葡萄糖激酶（GCK）具有21%的同源性。在功能上，与人类GCK相比，金黄色葡萄球菌glkA显示出更高的G-6-P形成速率，这可能在发病机制中起重要作用。
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