Enzymes dehalogenases

Figure C2.7.8. Catalytic cycle indicating the working of tire enzyme haloalkane dehalogenase [11],...

Nucleophilic substitution is one of a variety of mechanisms by which living systems detoxify halogenated organic compounds introduced into the environment Enzymes that catalyze these reactions are known as haloalkane dehalogenases The hydrolysis of 1 2 dichloroethane to 2 chloroethanol for example is a biological nude ophilic substitution catalyzed by a dehalogenase... 

The reversibility of halohydrin dehalogenase-catalyzed reactions has been used for the regioselective epoxide-opening with nonnatural nucleophiles (an example is given in Scheme 10.34) [133]. The stereoselectivity of the enzyme results in the resolution of the racemic substrate. At the same time, the regioselectivity imposed by the active site geometry yields the anti-Markovnikov product. [128]... 

Ring opening of epoxides with nucleophiles other than water (Cl, BrT I, NOz , CN ) can also be catalyzed by halohydrin dehalogenase enzymes (EC 3.8.1.5, also named haloalkane dehalogenase or haloalcohd dehalogenase) (Figure 6.74) [197]. 

Neumann A, A Siebert, T Trescher, S Reinhardt, G Wohlfarth, G Dickert (2002) Tetrachloroethene reductive dehalogenase of Dehalospirillum multivorans substrate specificity of the native enzyme and its cor-ronoid cofactor. Arch Microbiol 111 420-426. 

The haloalkane dehalogenase DhlA mechanism takes place in two consecutive Sn2 steps. In the first, the carboxylate moiety of the aspartate Aspl24, acting as a nucleophile on the carbon atom of DCE, displaces chloride anion which leads to formation of the enzyme-substrate intermediate (Equation 11.86). That intermediate is hydrolyzed by water in the subsequent step. The experimentally determined chlorine kinetic isotope effect for 1-chlorobutane, the slow substrate, is k(35Cl)/k(37Cl) = 1.0066 0.0004 and should correspond to the intrinsic isotope effect for the dehalogenation step. While the reported experimental value for DCE hydrolysis is smaller, it becomes practically the same when corrected for the intramolecular chlorine kinetic isotope effect (a consequence of the two identical chlorine labels in DCE). 

Amino acid sequence relationships have suggested a number of HYL families based on percent identity, enzymes with >40% identity belonging to the same family [48]. Families so identified include the mammalian microsomal EH (HYL1), the mammalian cytosolic EH (HYL2), the plant cytosolic EH (HYL3), and bacterial C-X bond hydrolases (haloacid dehydrogenases, HAD, and haloalkane dehalogenases, HLD). 

Yang et al reeently reported on the meehanism of 4-chlorobenzoyl eoenzyme A dehalogenase, an enzyme that catalyzes the hydrolytic dehalogenation of 4-chlo-robenzoyl coenzyme A (4-CBA-CoA) to form 4-hydro-xybenzoyl coenzyme A (4-HBA-CoA). The mechanism involves attack of an active site carboxylate at C4 of the substrate benzoyl ring to form a Meisenheimer complex (shown above). Loss of chloride ion from this intermediate then forms an arylated enzyme intermediate that is hydrolyzed to free enzyme plus 4-HBA-CoA by the addition of water at the acyl carbon. In later work, Taylor et al examined the activation of the 4-CB A-CoA toward nucleophilic attack by the active site carboxylate group. 

Scheme 6.1 Active sites of enzymes employing a double hydrogen-bonding motif for substrate coordination and activation in various biochemical transformations Haloalcohol dehalogenase (1), formate dehydrogenase (2), and serine protease (3).

Chemical mutagenesis produced a strain in which the S-CPA dehalogenase had been completely eliminated so that cheaper whole P. piitida cells could be used for the bioconversion, rather than more expensive extracted enzyme. 

Spray-dried cells have a stable enzyme activity enabling easy storage and transport of the dehalogenase biocatalyst. 

Whole cells are used in stirred tanks with pH control, producing fS )-2-chloropropanoic acid in 50% yield from the racemate (0.3 M) with an enantiomeric excess of over 95%. This approach was selected in preference to other methods of resolution such as acylation of the racemate and then stereoselective hydrolysis. The dehalogenase enzyme is specific for substrates with a carboxyl group and a 2-chloro or bromo substituent. No cofactor or metal ion is required and reaction involves an inversion of configuration. 

The dehalogenase activity of dehydrated cells of R. erythropolis, for both chlorinated and brominated compounds, increases with the carbon chain length of the compound (up to 1-chlorohexane and 1-bromohexane). The opposite has been reported for the aqueous phase for both the isolated enzyme [54] and resting cells [14]. Eliminating solubilization requirements also clearly improves rates of... 

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