Yew Wen Shan, Fedorov Alexander A, Fedorov Elena V, Almo Steven C, Gerlt John A
Departments of Biochemistry and Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.
Biochemistry. 2007 Aug 21;46(33):9564-77. doi: 10.1021/bi7008882. Epub 2007 Jul 25.
We assigned l-talarate dehydratase (TalrD) and galactarate dehydratase (GalrD) functions to a group of orthologous proteins in the mechanistically diverse enolase superfamily, focusing our characterization on the protein encoded by the Salmonella typhimurium LT2 genome (GI:16766982; STM3697). Like the homologous mandelate racemase, l-fuconate dehydratase, and d-tartrate dehydratase, the active site of TalrD/GalrD contains a general acid/base Lys 197 at the end of the second beta-strand in the (beta/alpha)7beta-barrel domain, Asp 226, Glu 252, and Glu 278 as ligands for the essential Mg2+ at the ends of the third, fourth, and fifth beta-strands, a general acid/base His 328-Asp 301 dyad at the ends of the seventh and sixth beta-strands, and an electrophilic Glu 348 at the end of the eighth beta-strand. We discovered the function of STM3697 by screening a library of acid sugars; it catalyzes the efficient dehydration of both l-talarate (kcat = 2.1 s-1, kcat/Km = 9.1 x 10(3) M-1 s-1) and galactarate (kcat = 3.5 s-1, kcat/Km = 1.1 x 10(4) M-1 s-1). Because l-talarate is a previously unknown metabolite, we demonstrated that S. typhimurium LT2 can utilize l-talarate as carbon source. Insertional disruption of the gene encoding STM3697 abolishes this phenotype; this disruption also diminishes, but does not eliminate, the ability of the organism to utilize galactarate as carbon source. The dehydration of l-talarate is accompanied by competing epimerization to galactarate; little epimerization to l-talarate is observed in the dehydration of galactarate. On the basis of (1) structures of the wild type enzyme complexed with l-lyxarohydroxamate, an analogue of the enolate intermediate, and of the K197A mutant complexed with l-glucarate, a substrate for exchange of the alpha-proton, and (2) incorporation of solvent deuterium into galactarate in competition with dehydration, we conclude that Lys 197 functions as the galactarate-specific base and His 328 functions as the l-talarate-specific base. The epimerization of l-talarate to galactarate that competes with dehydration can be rationalized by partitioning of the enolate intermediate between dehydration (departure of the 3-OH group catalyzed by the conjugate acid of His 328) and epimerization (protonation on C2 by the conjugate acid of Lys 197). The promiscuous catalytic activities discovered for STM3697 highlight the evolutionary potential of a "conserved" active site architecture.
我们将L - 塔罗糖酸脱水酶（TalrD）和半乳糖二酸脱水酶（GalrD）的功能赋予了机制多样的烯醇酶超家族中的一组直系同源蛋白，我们的表征重点是鼠伤寒沙门氏菌LT2基因组（GI:16766982；STM3697）编码的蛋白。与同源扁桃酸消旋酶、L - 岩藻糖酸脱水酶和D - 酒石酸脱水酶一样，TalrD / GalrD的活性位点在（β/α）7β桶结构域的第二条β链末端含有一个通用酸/碱赖氨酸197，在第三条、第四条和第五条β链末端含有作为必需Mg2 +配体的天冬氨酸226、谷氨酸252和谷氨酸278，在第七条和第六条β链末端含有一个通用酸/碱组氨酸328 - 天冬氨酸301二元组，在第八条β链末端含有一个亲电谷氨酸348。我们通过筛选酸性糖文库发现了STM3697的功能；它催化L - 塔罗糖酸（kcat = 2.1 s-1，kcat/Km = 9.1×10(3) M-1 s-1）和半乳糖二酸（kcat = 3.5 s-1，kcat/Km = 1.1×10(4) M-1 s-1）的高效脱水。由于L - 塔罗糖酸是一种先前未知的代谢物，我们证明了鼠伤寒沙门氏菌LT2可以利用L - 塔罗糖酸作为碳源。编码STM3697的基因的插入破坏消除了这种表型；这种破坏也降低了但没有消除该生物体利用半乳糖二酸作为碳源的能力。L - 塔罗糖酸的脱水伴随着向半乳糖二酸的竞争性差向异构化；在半乳糖二酸的脱水中未观察到向L - 塔罗糖酸的差向异构化。基于（1）与烯醇中间体类似物L - 来苏糖异羟肟酸复合以及与α - 质子交换底物L - 葡糖二酸复合的K197A突变体的野生型酶的结构，以及（2）溶剂氘与脱水竞争掺入半乳糖二酸，我们得出结论，赖氨酸197作为半乳糖二酸特异性碱基起作用，组氨酸328作为L - 塔罗糖酸特异性碱基起作用。与脱水竞争的L - 塔罗糖酸向半乳糖二酸的差向异构化可以通过烯醇中间体在脱水（由组氨酸328的共轭酸催化3 - OH基团离去）和差向异构化（由赖氨酸197的共轭酸在C2上质子化）之间的分配来解释。为STM3697发现的混杂催化活性突出了“保守”活性位点结构的进化潜力。
