Stereochemistry reveals the mechanism of bacterial alkane activation without oxygen

Abstract

The denitrifying bacterium strain HxN1 converts hexane and fumarate to 4- methyloctanyl-CoA. It was proposed that an initial adduct derived from fumarate and the hex-2-yl radical is converted to a CoA-thioester, (1'-methylpentyl)- succinyl-CoA 1, which rearranges to (2-methylhexyl)malonyl-CoA 2 by a mechanism similar to that of coenzyme B12-dependent radical enzyme methylmalonyl-CoA mutase. Decarboxylation of (2-methylhexyl)malonyl-CoA affords 4-methyloctanyl-CoA 3: To explore the stereochemistry of the mechanism of hexane degradation we have synthesised hexanes specifically labelled with deuterium: (2R,5R)-2,5- dideuteriohexane, (2S,5S)-2,5-dideuteriohexane, (2R,5S)-2,5-dideuteriohexane, 2,2,5,5-tetradeuteriohexane and 2,2-dideuteriohexane. This was achieved by tosylation of the relevant diol followed by reduction using LiAl2H4 as shown in example below: Analysis of products from the action of HxN1 on these labelled hexanes showed that the pro-S hydrogen is abstracted from C-2 of hexane with a primary kinetic isotope effect of ca. 3: (i) p-toluenesulfonyl chloride, pyridine in dichloromethane, 0 °C, 72 h; (Ts = p-toluenesulfonyl); (ii) LiAl2H4, tetraglyme, 120 °C, 2 h. iv To elucidate the configuration at the newly formed stereocenters, all four stereoisomers of (1-methylpentyl)succinate were synthesised as shown below, in order to use them as standards for comparison with metabolites from strain HxN1. Comparison using gas chromatography (GC) showed that, anaerobic growth of the bacterium strain HxN1 with n-hexane gives nearly equal amounts of (2R,1′R)- and (2S,1′R)-(1-methylpentyl)succinate, which are formed by the radical addition of the hydrocarbon to fumarate. As a result of these stereochemical studies, a new concerted mechanism has been postulated for the enzymatic reaction combining hexane with fumarate. The anaerobic degradation pathways of the environmentally relevant polycyclic aromatic hydrocarbons are largely unknown and therefore the final part of this thesis describes a study of the enzymatic de-aromatisation reactions involved in the degradation of naphthalene by the sulfate-reducing enrichment culture N47. This study required the synthesis of 1,4,4a,5,6,7,8,8a-octahydronaphthalene-2- carboxylic acid and 3,4,4a,5,6,7,8,8a-octahydronaphthalene-2-carboxylic acid, in order to check whether these compounds are intermediates in the degradation of naphthalene. Synthesis of these compounds was achieved using the method shown below: (i) H2SO4, MeOH, (ii) NaCN, H2O, RT, 5 h; (iii) 2,6-lutidine, SOCl2, Et2O, 60 °C, 12 h; (iv) H2SO4, H2O, 110 °C, 24 h. v HPLC, GCMS, NMR and UV/vis spectrum analysis suggested that 5,6,7,8- tetrahydro-2-NCoA (THNCoA) is reduced by two electrons rather than by four electrons as suggested, therefore affording one of the possible hexahydro- 2-naphthoyl-CoA (HHNCoA) isomers: