Cloning and Characterization of D-xylulose Kinase from Kocuria gwangalliensis Strain SJ2

D-Xylulose is phosphorylated to D-xylulose-5-phosphate by D-xylulose kinase before it enters glycolysis via the nonoxidative pentose phosphate pathway. A gene encoding a novel D-xylulose kinase (XK) from K. gwangalliensis strain SJ2 was sequenced and expressed in E. coli. The sequence of the isolated XK gene was 1,419 bp, encoding 472 amino acids. The XK protein was more closely related to the Arthrobacter phenanthrenivorans XK than to the Bifidobacterium catenulatum one, as reflected in the sequence identity (54.9% vs. 38.7%). The XK gene was subcloned into the pCold-II expression vector. The resulting plasmid was transformed into E. coli strain BL21 (DE3) cells and the expression of the recombinant XK protein was induced by the addition of IPTG. The resulting protein was expressed as a fusion protein of approximately 48 kDa containing a N-terminal six-histidine extension that was derived from the expression vector. The expressed protein was homogenized by affinity chromatography and showed enzymatic activity corresponding to D-xylulose kinase. XK enzyme kinetic studies with D-xylulose and ATP showed a Km of 250±20 μM and 1,300±50 μM, respectively. The results obtained from this study will provide a wider knowledge base for the characterization of D-xylulose kinase at the molecular level.

Kocuria gwangalliensis strain SJ2에서 유래된 D-xylulose kinase 유전자의 클로닝과 특성 연구

D-Xylulose는 nonoxidative pentose phosphate 경로를 통해 glycolysis 과정으로 들어가기 전에 D-xylulose kinase에 의해서 D-xylulose-5-phosphate로 인산화 된다. K. gwangalliensis strain SJ2로부터 D-xylulose kinase (XK)를 암호화하는 유전자는 E. coli를 이용하여 서열분석 및 발현 하였으며, XK 유전자의 염기서열 1,419 bp로 구성되어 있으며 463개의 아미노산 잔기를 암호화하고 있다. 분석결과를 통해 XK 유전자가 진화과정 동안 잘 보존되었음을 보여 주었다. XK 유전자의 발현을 위해 pCold-II 발현 벡터에 클로닝 하였으며 클로닝 된 플라스미드는 E. coli strain BL21 (DE3)에 형질전환 하여 IPTG를 이용해 발현을 유도하였다. 재조합 된 XK 단백질의 크기는 약 48 kDa이었다. 이 발현된 단백질은 affinity chromatography를 이용하여 정제하였으며 D-xylulose kinase에 따른 enzymatic activity를 분석하였다. D-xylulose와 ATP로 실행한 XK enzyme kinetic 연구는 각각 250±20 μM과 1,300±50 μM의 Km value를 보였다. 본 연구를 통해 얻어진 결과는 분자적 수준에서 D-xylulose kinase의 특성연구의 보다 넓은 지식적 기초를 제공할 것으로 사료된다.