Evolution of Enzymatic Activities in the Enolase Superfamily: D-Mannonate Dehydratase from Novosphingobium aromaticivorans

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• 作者：John F. Rakus ; Alexander A. Fedorov ; Elena V. Fedorov ; Margaret E. Glasner ; Jacob E. Vick ; Patricia C. Babbitt ; Steven C. Almo ; John A. Gerlt
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：November 13, 2007
• 年：2007
• 卷：46
• 期：45
• 页码：12896 - 12908
• 全文大小：923K
• 年卷期：v.46,no.45(November 13, 2007)
• ISSN：1520-4995

文摘

The D-mannonate dehydratase (ManD) function was assigned to a group of orthologous proteinsin the mechanistically diverse enolase superfamily by screening a library of acid sugars. Structures of thewild type ManD from Novosphingobium aromaticivorans were determined at pH 7.5 in the presence ofMg²⁺ and also in the presence of Mg²⁺ and the 2-keto-3-keto-D-gluconate dehydration product; the structureof the catalytically active K271E mutant was determined at pH 5.5 in the presence of the D-mannonatesubstrate. As previously observed in the structures of other members of the enolase superfamily, ManDcontains two domains, an N-terminal + capping domain and a (/)₇-barrel domain. The barrel domaincontains the ligands for the essential Mg²⁺, Asp 210, Glu 236, and Glu 262, at the ends of the third,fourth, and fifth -strands of the barrel domain, respectively. However, the barrel domain lacks both theLys acid/base catalyst at the end of the second -strand and the His-Asp dyad acid/base catalyst at theends of the seventh and sixth -strands, respectively, that are found in many members of the superfamily.Instead, a hydrogen-bonded dyad of Tyr 159 in a loop following the second -strand and Arg 147 at theend of the second -strand are positioned to initiate the reaction by abstraction of the 2-proton. Both Tyr159 and His 212, at the end of the third -strand, are positioned to facilitate both syn-dehydration andketonization of the resulting enol intermediate to yield the 2-keto-3-keto-D-gluconate product with theobserved retention of configuration. The identities and locations of these acid/base catalysts as well as ofcationic amino acid residues that stabilize the enolate anion intermediate define a new structural strategyfor catalysis (subgroup) in the mechanistically diverse enolase superfamily. With these differences, weprovide additional evidence that the ligands for the essential Mg²⁺ are the only conserved residues in theenolase superfamily, establishing the primary functional importance of the Mg²⁺-assisted strategy forstabilizing the enolate anion intermediate.