Divergence of Structure and Function in the Haloacid Dehalogenase Enzyme Superfamily: Bacteroides thetaiotaomicron BT2127 Is an Inorganic Pyrophosphatase
Divergence of Structure and Function in the Haloacid Dehalogenase Enzyme Superfamily: Bacteroides thetaiotaomicron BT2127 Is an Inorganic Pyrophosphatase
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DOI：
10.1021/bi201181q
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发表时间：
2011-10-18
期刊：
影响因子：
2.9
通讯作者：
Dunaway-Mariano, Debra
中科院分区：
文献类型：
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作者：
Huang, Hua;Patskovsky, Yury;Dunaway-Mariano, Debra
蛋白质序列信息的爆炸性增长要求当前的功能分配策略演变为基于计算的功能预测来补充实验方法。这就需要在以特异性决定因素的形式识别序列标记和更知情的同源定义的基础上制定策略。在这里，我们利用生物信息学-结构-机制集成的方法对未知的卤代烷酸脱卤酶超家族成员BT2127(UniProt登录码Q8A5 V9)进行了功能指定。BT2127的底物专一性和稳态速率常数(焦磷酸的k(CAT)/k(M)值类似于1×10(5)M-1 S(-1))，以及基因背景，支持指定的体内无机焦磷酸酶的功能。对野生型BT2127和通过定点突变产生的几个变体的X射线结构分析表明，底物识别在一定程度上是基于帽结构域(特别是由Tyr76和Glu47残基)施加的活性位点空间限制。结构导向定点突变与突变酶的动力学分析相结合，确定了催化、底物结合和结构域结合所需的残基。在此结构功能分析的基础上，以催化残基Asp11、Asp13、Thr113和Lys147以及金属结合残基Asp171、Asn172和Glu47为标记，通过序列搜索确认BT2127同源物。这一生物信息学分析表明，BT2127同源基因的生物学范围仅限于拟杆菌/氯杆菌门。BT2127及其最接近的同源物-β-磷酸葡萄糖变位酶差异的关键结构决定因素控制着离开基团的大小(磷酸和葡萄糖磷酸)以及天冬氨酸/碱在开放构象和闭合构象中的位置。HADSF焦磷酸酶代表了细菌焦磷酸酶的第三种机制和折叠型。
The explosion of protein sequence information requires that current strategies for function assignment evolve to complement experimental approaches with computationally based function prediction. This necessitates the development of strategies based on the identification of sequence markers in the form of specificity determinants and a more informed definition of orthologues. Herein, we have undertaken the function assignment of the unknown haloalkanoate dehalogenase superfamily member BT2127 (Uniprot accession code Q8A5 V9) from Bacteroides thetaiotaomicron using an integrated bioinformatics-structure-mechanism approach. The substrate specificity profile and steady-state rate constants of BT2127 (with a k(cat)/k(m) value for pyrophosphate of similar to 1 x 10(5) M-1 s(-1)), together with the gene context, support the assigned in vivo function as an inorganic pyrophosphatase. The X-ray structural analysis of wild-type BT2127 and several variants generated by site-directed mutagenesis shows that substrate discrimination is based, in part, on active site space restrictions imposed by the cap domain (specifically by residues Tyr76 and Glu47). Structure-guided site-directed mutagenesis coupled with kinetic analysis of the mutant enzymes identified the residues required for catalysis, substrate binding, and domain domain association. On the basis of this structure function analysis, the catalytic residues Asp11, Asp13, Thr113, and Lys147 as well the metal binding residues Asp171, Asn172, and Glu47 were used as markers to confirm BT2127 orthologues identified via sequence searches. This bioinformatic analysis demonstrated that the biological range of BT2127 orthologue is restricted to the phylum Bacteroidetes/Chlorobi. The key structural determinants in the divergence of BT2127 and its closest homologue, beta-phosphoglucomutase, control the leaving group size (phosphate vs glucose phosphate) and the position of the Asp acid/base in the open versus closed conformations. HADSF pyrophosphatases represent a third mechanistic and fold type for bacterial pyrophosphatases.