Acinetobacter strain IVS-B aerobically grows on isovalerate as sole carbon and energy source. Isovalerate is metabolised via isovaleryl-CoA, an intermediate of the oxidative (S)-leucine degradation pathway. A 3-methylglutaconyl-CoA hydratase (EC 4.2.1.18) was purified 65-fold to apparent homogeneity from cell-free extracts of isovalerate-grown cells of Acinetobacter strain IVS-B. The enzyme was found to be a homotetramer (115.2 kDa) composed of four identical subunits of 28.8 kDa not containing any cofactors. The enzyme was shown to catalyse the hydration of (E)-glutaconyl-CoA (k (cat)=18 s(-1), K (m)=40 microM) and the dehydration of (S)-3-hydroxyglutaryl-CoA (k (cat)=13 s(-1), K (m)=52 microM), albeit with somewhat lower catalytic efficiencies as compared to the 3-methyl derivatives, 3-methylglutaconyl-CoA (k (cat)=138 s(-1), K (m)=14 microM) and (S)-3-hydroxy-3-methylglutaryl-CoA (k (cat)=60 s(-1), K (m)=36 microM). Thus, the mechanistically simple syn-addition of water to the (E)-isomer of 3-methylglutaconyl-CoA of the leucine degradative pathway leading to the common intermediate (S)-3-hydroxy-3-methylglutaryl-CoA was assigned as the major physiological role to this enzyme. The amino acid sequence of 3-methylglutaconyl-CoA hydratase from Acinetobacter sp. was found to be related to over 100 prokaryotic enoyl-CoA hydratases (up to 50% identity), possibly all being 3-methylglutaconyl-CoA hydratases.