The atuRABCDEFGH gene cluster is essential for acyclic terpene utilization (Atu) in Pseudomonas aeruginosa. The biochemical functions of most Atu proteins have not been experimentally verified; exceptions are AtuC/AtuF, which constitute the two subunits of geranyl-CoA carboxylase, the key enzyme of the Atu pathway. In this study we investigated the biochemical function of AtuD and of the PA1535 gene product, a protein related to AtuD in amino acid sequence. 2D gel electrophoresis showed that AtuD and the PA1535 protein were specifically expressed in cells grown on acyclic terpenes but were absent in isovalerate- or succinate-grown cells. Mutant analysis indicated that AtuD but not the product of PA1535 is essential for acyclic terpene utilization. AtuD and PA1535 gene product were expressed in recombinant Escherichia coli and purified to homogeneity. Purified AtuD showed citronellyl-CoA dehydrogenase activity (V(max) 850 mU mg(-1)) and high affinity to citronellyl-CoA (K(m) 1.6 microM). AtuD was inactive with octanoyl-CoA, 5-methylhex-4-enoyl-CoA or isovaleryl-CoA. Purified PA1535 gene product revealed high citronellyl-CoA dehydrogenase activity (V(max) 2450 mU mg(-1)) but had significantly lower affinity than AtuD to citronellyl-CoA (K(m) 18 microM). Purified PA1535 protein additionally utilized octanoyl-CoA as substrate (V(max), 610 mU mg(-1); K(m) 130 microM). To our knowledge AtuD is the first acyl-CoA dehydrogenase with a documented substrate specificity for terpenoid molecule structure and is essential for a functional Atu pathway. Potential other terpenoid-CoA dehydrogenases were found in the genomes of Pseudomonas citronellolis, Marinobacter aquaeolei and Hahella chejuensis but were absent in non-acyclic terpene-utilizing bacteria.