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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Directed evolution approach to a structural genomics project: Rv2002 from Mycobacterium tuberculosis.
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Directed evolution approach to a structural genomics project: Rv2002 from Mycobacterium tuberculosis.

机译：针对结构基因组学项目的定向进化方法：来自结核分枝杆菌的Rv2002。

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摘要

One of the serious bottlenecks in structural genomics projects is overexpression of the target proteins in soluble form. We have applied the directed evolution technique and prepared soluble mutants of the Mycobacterium tuberculosis Rv2002 gene product, the wild type of which had been expressed as inclusion bodies in Escherichia coli. A triple mutant I6TV47MT69K (Rv2002-M3) was chosen for structural and functional characterizations. Enzymatic assays indicate that the Rv2002-M3 protein has a high catalytic activity as a NADH-dependent 3alpha, 20beta-hydroxysteroid dehydrogenase. We have determined the crystal structures of a binary complex with NAD(+) and a ternary complex with androsterone and NADH. The structure reveals that Asp-38 determines the cofactor specificity. The catalytic site includes the triad Ser-140Tyr-153Lys-157. Additionally, it has an unusual feature, Glu-142. Enzymatic assays of the E142A mutant of Rv2002-M3 indicate that Glu-142 reverses the effect of Lys-157 in influencing the pKa of Tyr-153. This study suggests that the Rv2002 gene product is a unique member of the SDR family and is likely to be involved in steroid metabolism in M. tuberculosis. Our work demonstrates the power of the directed evolution technique as a general way of overcoming the difficulties in overexpressing the target proteins in soluble form.
机译：结构基因组学计划中的严重瓶颈之一是可溶性蛋白形式的靶蛋白过表达。我们已经应用了定向进化技术，并制备了结核分枝杆菌Rv2002基因产物的可溶性突变体，其野生型已在大肠杆菌中表达为包涵体。选择了三重突变体I6TV47MT69K（Rv2002-M3）进行结构和功能表征。酶法测定表明，Rv2002-M3蛋白作为依赖NADH的3alpha，20beta-羟类固醇脱氢酶具有很高的催化活性。我们已经确定了具有NAD（+）的二元配合物和具有雄甾酮和NADH的三元配合物的晶体结构。结构表明Asp-38决定了辅因子的特异性。催化部位包括三联体Ser-140Tyr-153Lys-157。此外，它还有一个不寻常的功能Glu-142。 Rv2002-M3的E142A突变体的酶促测定表明，Glu-142逆转了Lys-157对Tyr-153的pKa的影响。这项研究表明，Rv2002基因产物是SDR家族的独特成员，可能参与了结核分枝杆菌的类固醇代谢。我们的工作证明了定向进化技术作为克服以可溶形式过量表达靶蛋白的困难的一般方法的力量。

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