الفهرس 
Haloacrylate hydratase, a new class of flavoenzymeOnly 14 pages are availabe for public view
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Abstract
The present study has been carried out to characterize an enzyme involved in the metabolism of 2-chloroacrylic acid (2-CAA) in Pseudomonas sp. YL.. In addition, we constructed an enzymatic system to produce (S)-2-chloropropionate. The comparison of the proteins produced in 2-CAA-grown cells and lactate-grown cells resulted in the identification of a protein inducibly expressed in 2-CAA-grown cells. This enzyme (named as 2-haloacrylate hydratase) catalyzes the conversion of 2-CAA and 2-bromoacrylate to pyruvate, and the reduced form of FAD is required for catalysis. In the presence of H218O, [18O]-pyruvate was produced indicating that this enzyme catalyzes the hydration of 2-chloroacrylate to form pyruvate and HCl, a catalysis which involves no net change in redox state of the coenzyme or substrate. The reduced form of FAD was found to be generated by the intrinsic NADH-dependent FAD reductase activity of this enzyme. The data shown in Chapter III strongly support a crucial catalytic role of the reduced flavin in the hydration catalyzed by 2-haloacrylate hydratase. The generation of FADsq in a 2-CAA-dependent manner and its stabilization during photoreduction suggest that FADsq is produced as an intermediate during the reaction. The restoration of the activity with 1-deazaFAD, in addition to the formation of a radical all together lead us to propose a radical-mediated mechanism in which FADH2 may donate one electron to the substrate to produce a radical intermediate, which facilitates protonation at the C-3 position and subsequently a nucleophilic attack would happen to produce 2-chloro-2-hydroxypropionate. The recombinant E. coli cells expressing both 2-haloacrylate reductase and glucose dehydrogenase were used the production of (S)-2-chloropropionate with 89.9% yield.