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J. Biol. Chem., Vol. 279, Issue 3, 2242-2253, January 16, 2004

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The Crystal Structures of Klebsiella pneumoniae Acetolactate Synthase with Enzyme-bound Cofactor and with an Unusual Intermediate^*

Siew Siew Pang, Ronald G. Duggleby, Richard L. Schowen¶, and Luke W. Guddat

From the Department of Biochemistry and Molecular Biology, the University of Queensland, Brisbane, Queensland 4072, Australia and the ^¶Department of Chemistry, the University of Kansas, Lawrence, Kansas 66045

Acetohydroxyacid synthase (AHAS) and acetolactate synthase (ALS) are thiamine diphosphate (ThDP)-dependent enzymes that catalyze the decarboxylation of pyruvate to give a cofactor-bound hydroxyethyl group, which is transferred to a second molecule of pyruvate to give 2-acetolactate. AHAS is found in plants, fungi, and bacteria, is involved in the biosynthesis of the branched-chain amino acids, and contains non-catalytic FAD. ALS is found only in some bacteria, is a catabolic enzyme required for the butanediol fermentation, and does not contain FAD. Here we report the 2.3-Å crystal structure of Klebsiella pneumoniae ALS. The overall structure is similar to AHAS except for a groove that accommodates FAD in AHAS, which is filled with amino acid side chains in ALS. The ThDP cofactor has an unusual conformation that is unprecedented among the 26 known three-dimensional structures of nine ThDP-dependent enzymes, including AHAS. This conformation suggests a novel mechanism for ALS. A second structure, at 2.0 Å, is described in which the enzyme is trapped halfway through the catalytic cycle so that it contains the hydroxyethyl intermediate bound to ThDP. The cofactor has a tricyclic structure that has not been observed previously in any ThDP-dependent enzyme, although similar structures are well known for free thiamine. This structure is consistent with our proposed mechanism and probably results from an intramolecular proton transfer within a tricyclic carbanion that is the true reaction intermediate. Modeling of the second molecule of pyruvate into the active site of the enzyme with the bound intermediate is consistent with the stereochemistry and specificity of ALS.

Received for publication, April 17, 2003 , and in revised form, October 10, 2003.

^* This work was supported by Grant A00105313 (to R. G. D. and L. W. G.) by the Australian Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 61-7-3365-4615; Fax: 61-7-3365-4699; E-mail: Ronald.Duggleby{at}mailbox.uq.edu.au.

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