Sousa Sílvia A, Moreira Leonilde M, Leitão Jorge H
IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Lisbon, Portugal.
Appl Microbiol Biotechnol. 2008 Oct;80(6):1015-22. doi: 10.1007/s00253-008-1612-1. Epub 2008 Jul 31.
The bceA(J) gene from the cystic fibrosis isolate Burkholderia cenocepacia J2315 encodes a 56-kDa bifunctional protein, with phosphomannose isomerase (PMI) and guanosine diphosphate (GDP)-mannose pyrophosphorylase (GMP) activities, a new member of the poorly characterised type II PMI class of proteins. Due to the lack of homology between the type II PMIs and the human PMI, this class of proteins are being regarded as interesting potential targets to develop new antimicrobials. The BceA(J) protein conserves the four typical motifs of type II PMIs: the pyrophosphorylase signature, the GMP active site, the PMI active site and the zinc-binding motif. After overproduction of BceA(J) by Escherichia coli as a histidine tag derivative, the protein was purified to homogeneity by affinity chromatography. The GMP activity is dependent on the presence of Mg(2+) or Ca(2+) as cofactors, while the PMI activity uses a broader range of divalent ions, in the order of activation Mg(2+) > Ca(2+) > Mn(2+) > Co(2+) > Ni(2+). The kinetic parameters K(m), V(max) and K(cat)/K(m) for the PMI and GMP activities were determined. Results suggest that the enzyme favours the formation of GDP-mannose instead of mannose catabolism, thus channelling precursors to the formation of glycoconjugates.
来自囊性纤维化分离株洋葱伯克霍尔德菌J2315的bceA(J)基因编码一种56 kDa的双功能蛋白，具有磷酸甘露糖异构酶（PMI）和鸟苷二磷酸（GDP）-甘露糖焦磷酸化酶（GMP）活性，是特征描述较少的II型PMI类蛋白的新成员。由于II型PMI与人PMI之间缺乏同源性，这类蛋白被视为开发新型抗菌药物的有趣潜在靶点。BceA(J)蛋白保留了II型PMI的四个典型基序：焦磷酸化酶特征序列、GMP活性位点、PMI活性位点和锌结合基序。大肠杆菌将BceA(J)作为组氨酸标签衍生物过量表达后，通过亲和层析将该蛋白纯化至同质。GMP活性依赖于Mg(2+)或Ca(2+)作为辅因子的存在，而PMI活性使用更广泛的二价离子，激活顺序为Mg(2+) > Ca(2+) > Mn(2+) > Co(2+) > Ni(2+)。测定了PMI和GMP活性的动力学参数K(m)、V(max)和K(cat)/K(m)。结果表明，该酶有利于GDP-甘露糖的形成而非甘露糖分解代谢，从而将前体导向糖缀合物的形成。
