Crystal Structure and Enzyme Kinetics of the (S)-Specific 1-Phenylethanol Dehydrogenase of the Denitrifying Bacterium Strain EbN1
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- 作者：H. Wolfgang Hö ; ffken ; Minh Duong ; Thomas Friedrich ; Michael Breuer ; Bernhard Hauer ; Richard Reinhardt ; Ralf Rabus ; and Johann Heider
- 刊名：Biochemistry
- 出版年：2006
- 出版时间：January 10, 2006
- 年：2006
- 卷：45
- 期：1
- 页码：82 - 93
- 全文大小：649K
- 年卷期：v.45,no.1(January 10, 2006)
- ISSN：1520-4995
文摘
(S)-1-Phenylethanol dehydrogenase (PED) from the denitrifying bacterium strain EbN1 catalyzesthe NAD+-dependent, stereospecific oxidation of (S)-1-phenylethanol to acetophenone and the biotechnologically interesting reverse reaction. This novel enzyme belongs to the short-chain alcohol dehydrogenase/aldehyde reductase family. The coding gene (ped) was heterologously expressed in Escherichiacoli and the purified protein was crystallized. The X-ray structures of the apo-form and the NAD+-boundform were solved at a resolution of 2.1 and 2.4 Å, respectively, revealing that the enzyme is a tetramerwith two types of hydrophobic dimerization interfaces, similar to 
beta2.gif" BORDER=0 ALIGN="middle">-oxoacyl-[acyl carrier protein] reductase(FabG) from E. coli. NAD+-binding is associated with a conformational shift of the substrate bindingloop of PED from a crystallographically unordered "open" to a more ordered "closed" form. Modelingthe substrate acetophenone into the active site revealed the structural prerequisites for the strongenantioselectivity of the enzyme and for the catalytic mechanism. Studies on the steady-state kinetics ofPED indicated a highly positive cooperativity of both catalytic directions with respect to the substrates.This is contrasted by the behavior of FabG. Moreover, PED exhibits extensive regulation on the enzymelevel, being inhibited by elevated concentrations of substrates and products, as well as the wrong enantiomerof 1-phenylethanol. These regulatory properties of PED are consistent with the presence of a putative"transmission module" between the subunits. This module consists of the C-terminal loops of all foursubunits, which form a special interconnected structural domain and mediate close contact of the subunits,and of a phenylalanine residue in each subunit that reaches out between substrate-binding loop andC-terminal domain of an adjacent subunit. These elements may transmit the substrate-induced conformational change of the substrate binding loop from one subunit to the others in the tetrameric complexand thus mediate the cooperative behavior of PED.
beta2.gif" BORDER=0 ALIGN="middle">-oxoacyl-[acyl carrier protein] reductase(FabG) from E. coli. NAD+-binding is associated with a conformational shift of the substrate bindingloop of PED from a crystallographically unordered "open" to a more ordered "closed" form. Modelingthe substrate acetophenone into the active site revealed the structural prerequisites for the strongenantioselectivity of the enzyme and for the catalytic mechanism. Studies on the steady-state kinetics ofPED indicated a highly positive cooperativity of both catalytic directions with respect to the substrates.This is contrasted by the behavior of FabG. Moreover, PED exhibits extensive regulation on the enzymelevel, being inhibited by elevated concentrations of substrates and products, as well as the wrong enantiomerof 1-phenylethanol. These regulatory properties of PED are consistent with the presence of a putative"transmission module" between the subunits. This module consists of the C-terminal loops of all foursubunits, which form a special interconnected structural domain and mediate close contact of the subunits,and of a phenylalanine residue in each subunit that reaches out between substrate-binding loop andC-terminal domain of an adjacent subunit. These elements may transmit the substrate-induced conformational change of the substrate binding loop from one subunit to the others in the tetrameric complexand thus mediate the cooperative behavior of PED.