Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Apartado 127, P-2781-901 Oeiras, Portugal.
J Bacteriol. 2011 Aug;193(15):3978-87. doi: 10.1128/JB.01076-10. Epub 2011 May 20.
Members of the Burkholderia cepacia complex (BCC) are serious respiratory pathogens in immunocompromised individuals and in patients with cystic fibrosis (CF). They are exceptionally resistant to many antimicrobial agents and have the capacity to spread between patients, leading to a decline in lung function and necrotizing pneumonia. BCC members often express a mucoid phenotype associated with the secretion of the exopolysaccharide (EPS) cepacian. There is much evidence supporting the fact that cepacian is a major virulence factor of BCC. UDP-glucose dehydrogenase (UGD) is responsible for the NAD-dependent 2-fold oxidation of UDP-glucose (UDP-Glc) to UDP-glucuronic acid (UDP-GlcA), which is a key step in cepacian biosynthesis. Here, we report the structure of BceC, determined at 1.75-Å resolution. Mutagenic studies were performed on the active sites of UGDs, and together with the crystallographic structures, they elucidate the molecular mechanism of this family of sugar nucleotide-modifying enzymes. Superposition with the structures of human and other bacterial UGDs showed an active site with high structural homology. This family contains a strictly conserved tyrosine residue (Y10 in BceC; shown in italics) within the glycine-rich motif (GXGYXG) of its N-terminal Rossmann-like domain. We constructed several BceC Y10 mutants, revealing only residual dehydrogenase activity and thus highlighting the importance of this conserved residue in the catalytic activity of BceC. Based on the literature of the UGD/GMD nucleotide sugar 6-dehydrogenase family and the kinetic and structural data we obtained for BceC, we determined Y10 as a key catalytic residue in a UGD rate-determining step, the final hydrolysis of the enzymatic thioester intermediate.
洋葱伯克霍尔德菌复合体（BCC）成员是免疫功能低下个体和囊性纤维化（CF）患者的严重呼吸道病原体。它们对许多抗菌药物具有极强的耐药性，并且能够在患者之间传播，导致肺功能下降和坏死性肺炎。BCC 成员通常表现出与外多糖（EPS）cepacian 分泌相关的粘液表型。有大量证据支持 cepacian 是 BCC 的主要毒力因子这一事实。UDP-葡萄糖脱氢酶（UGD）负责 NAD 依赖性 2 倍氧化 UDP-葡萄糖（UDP-Glc）为 UDP-葡萄糖醛酸（UDP-GlcA），这是 cepacian 生物合成的关键步骤。在这里，我们报道了 BceC 的结构，其分辨率为 1.75 Å。在 UGD 的活性部位进行了诱变研究，结合晶体结构，阐明了这一家族糖核苷酸修饰酶的分子机制。与人和其他细菌 UGD 的结构叠加显示出具有高结构同源性的活性部位。该家族在其 N 端 Rossmann 样结构域的甘氨酸丰富基序（GXGYXG）中包含一个严格保守的酪氨酸残基（BceC 中的 Y10；用斜体显示）。我们构建了几个 BceC Y10 突变体，仅显示出残留的脱氢酶活性，从而突出了该保守残基在 BceC 催化活性中的重要性。基于 UGD/GMD 核苷酸糖 6-脱氢酶家族的文献以及我们获得的 BceC 的动力学和结构数据，我们确定 Y10 是 UGD 速率决定步骤（酶硫酯中间体的最终水解）中的关键催化残基。
