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J. Biol. Chem., Vol. 278, Issue 46, 45094-45100, November 14, 2003

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Catalytic Mechanism of the Haloalkane Dehalogenase LinB from Sphingomonas paucimobilis UT26^*

Zbynk Prokop, Marta Monincová, Radka Chaloupková, Martin Klvaa, Yuji Nagata¶, Dick B. Janssen||, and Jií Damborsk

From the National Centre for Biomolecular Research, Masaryk University, Kotlarska 2, 611 37 Brno, Czech Republic, the ^¶Department of Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Sendai, 980-8577, Japan, and the ^||University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands

Haloalkane dehalogenases are bacterial enzymes capable of carbon-halogen bond cleavage in halogenated compounds. To obtain insights into the mechanism of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB), we studied the steady-state and presteady-state kinetics of the conversion of the substrates 1-chlorohexane, chlorocyclohexane, and bromocyclohexane. The results lead to a proposal of a minimal kinetic mechanism consisting of three main steps: (i) substrate binding, (ii) cleavage of the carbon-halogen bond with simultaneous formation of an alkyl-enzyme intermediate, and (iii) hydrolysis of the alkyl-enzyme intermediate. Release of both products, halide and alcohol, is a fast process that was not included in the reaction mechanism as a distinct step. Comparison of the kinetic mechanism of LinB with that of haloalkane dehalogenase DhlA from Xantobacter autotrophicus GJ10 and the haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 shows that the overall mechanisms are similar. The main difference is in the rate-limiting step, which is hydrolysis of the alkylenzyme intermediate in LinB, halide release in DhlA, and liberation of an alcohol in DhaA. The occurrence of different rate-limiting steps for three enzymes that belong to the same protein family indicates that extrapolation of this important catalytic property from one enzyme to another can be misleading even for evolutionary closely related proteins. The differences in the rate-limiting step were related to: (i) number and size of the entrance tunnels, (ii) protein flexibility, and (iii) composition of the halide-stabilizing active site residues based on comparison of protein structures.

Received for publication, July 2, 2003 , and in revised form, August 25, 2003.

^* This work was supported by Grant LN00A016 from the Czech Ministry of Education. 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.

Recipient of a scholarship from the Federation of European Microbiological Societies. To whom correspondence should be addressed. Fax: 420-5-41129506; E-mail: zbynek{at}chemi.muni.cz.

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