Pseudomonas species strain SBV1 can rapidly grow on medium containing beta-valine as a sole nitrogen source. The tertiary amine feature of beta-valine prevents direct deamination reactions catalyzed by aminotransferases, amino acid dehydrogenases, and amino acid oxidases. However, lyase- or aminomutase-mediated conversions would be possible. To identify enzymes involved in the degradation of beta-valine, a PsSBV1 gene library was prepared and used to complement the beta-valine growth deficiency of a closely related Pseudomonas strain. This resulted in the identification of a gene encoding beta-valinyl-coenzyme A ligase (BvaA) and two genes encoding beta-valinyl-CoA ammonia lyases (BvaB1 and BvaB2). The BvaA protein demonstrated high sequence identity to several known phenylacetate CoA ligases. Purified BvaA enzyme did not convert phenyl acetic acid but was able to activate beta-valine in an adenosine triphosphate (ATP)- and CoA-dependent manner. The substrate range of the enzyme appears to be narrow, converting only beta-valine and to a lesser extent, 3-aminobutyrate and beta-alanine. Characterization of BvaB1 and BvaB2 revealed that both enzymes were able to deaminate beta-valinyl-CoA to produce 3-methylcrotonyl-CoA, a common intermediate in the leucine degradation pathway. Interestingly, BvaB1 and BvaB2 demonstrated no significant sequence identity to known CoA-dependent ammonia lyases, suggesting they belong to a new family of enzymes. BLAST searches revealed that BvaB1 and BvaB2 show high sequence identity to each other and to several enoyl-CoA hydratases, a class of enzymes that catalyze a similar reaction with water instead of amine as the leaving group.