2-Hydroxyglutaryl-CoA dehydratase from Clostridium symbiosum

Component D (HgdAB) of 2-hydroxyglutaryl-CoA dehydratase from Clostridium s ymbiosum was purified to homogeneity. It is able to use component A from Ac idaminococcus fermentans (HgdC) to initiate catalysis together with ATP, Mg 2+ and a strong reducing agent such as Ti(III) citrate. Component D from C. symbiosum has a 6 x higher specific activity compared with that from A. fe rmentans and contains a second [4Fe-4S] cluster but the same amount of ribo flavin 5'-phosphate (1.0 per heterodimeric enzyme, m = 100 kDa). Mossbauer spectroscopy revealed symmetric cube-type structures of the two [4Fe-4S](2) clusters. EPR spectroscopy showed the resistance of the clusters to reduc ing agents, but detected a sharp signal at g = 2.004 probably due to a stab ilized flavin semiquinone, Three genes from C. symbiosum coding for compone nts D (hgdA and hgdB) and A (hgdC) were cloned and sequenced. Primer extens ion experiments indicated that the genes are transcribed in the order hgdCA B from an operon only half the size of that from A. fermentans. Sequence co mparisons detected a close relationship to the dehydratase system from A. f ermentans and HgdA from Fusobacterium nucleatum, as well as to putative pro teins of unknown function from Archaeoglobus fulgidus. Lower, but significa nt, identities were found with putative enzymes from several methanogenic A rchaea and Escherichia coli, as well as with the mechanistically related be nzoyl-CoA reductases from the Proteobacteria Rhodopseudomonas palustris and Thauera aromatica.