Biodehalogenation: Oxidative and Hydrolytic Pathways in the Transformations of Acetonitrile, Chloroacetonitrile, Chloroacetic Acid, and Chloroacetamide by Methylosinus trichosporium OB-3b

Abstract

Resting cell suspensions of the soil methylotroph Methylosinus trichosporium OB-3b rapidly metabolize acetonitrile (ACN) and chloroacetonitrile (CCN) to bicarbonate. Formate, a natural substrate for this organism, is a major metabolite in both cases. The first step in these processes, established with 13C-labeled substrates and NMR analysis, is an oxygen insertion into the C−H bond to yield the corresponding cyanohydrins. With acetonitrile, the loss of HCN from the initially formed cyanohydrin (HOCH2CN) produces formaldehyde. The latter is rapidly converted to formate and CO2. With chloroacetonitrile, the first formed cyanohydrin [HOCHCl(CN)] may lose HCN to produce formyl chloride or lose HCl to yield formyl nitrile. The generation of CO, in addition to formate, implicates formyl chloride and/or formyl nitrile as intermediates. With neither ACN nor CCN is the nitrile moiety directly attacked or hydrolyzed. In particular, the nitrile hydrolysis products from CCN, chloroacetamide (CAM), and chloroacetic acid (CA) are not detected. In addition, hydrolysis of the C−Cl moiety of CCN does not occur. In contrast, incubation of CAM and CA with this organism does result in the corresponding hydroxy amide and acid directly. The slower dehalogenation of the amide and acid entail a direct microbial hydroxylation of the C−Cl bond. Based upon this study and related work with methylene and ethylene halides, it may be concluded that in the series ClCH2X (X = Cl, Br, CN) oxygen insertion is the initial step in dehalogenation by this organism. However, in the series ClCH2Y (Y = CH2Cl, CO2H, CONH2), the dehalogenation is the result of a direct hydrolysis of the C−Cl bond. This organism also transforms cyanide ion, but incubations with K13CN resulted in no detectable products.