Identification and Characterization of D-Hydroxyproline Dehydrogenase and Δ1-Pyrroline-4-hydroxy-2-carboxylate Deaminase Involved in Novel L-Hydroxyproline Metabolism of Bacteria METABOLIC CONVERGENT EVOLUTION

L-Hydroxyproline (4-hydroxyproline) mainly exists in collagen, and most bacteria cannot metabolize this hydroxyamino acid. Pseudomonas putida and Pseudomonas aeruginosa convert L-hydroxyproline to alpha-ketoglutarate via four hypothetical enzymatic steps different from known mammalian pathways, but the molecular background is rather unclear. Here, we identified and characterized for the first time two novel enzymes, D-hydroxyproline dehydrogenase and Delta(1)-pyrroline-4-hydroxy-2-carboxylate (Pyr4H2C) deaminase, involved in this hypothetical pathway. These genes were clustered together with genes encoding other catalytic enzymes on the bacterial genomes. D-Hydroxyproline dehydrogenases from P. putida and P. aeruginosa were completely different from known bacterial proline dehydrogenases and showed similar high specificity for substrate (D-hydroxyproline) and some artificial electron acceptor( s). On the other hand, the former is a homomeric enzyme only containing FAD as a prosthetic group, whereas the latter is a novel heterododecameric structure consisting of three different subunits (alpha(4)beta(4)gamma(4)), and two FADs, FMN, and [2Fe-2S] iron-sulfur cluster were contained in alpha beta gamma of the heterotrimeric unit. These results suggested that the L-hydroxyproline pathway clearly evolved convergently in P. putida and P. aeruginosa. Pyr4H2C deaminase is a unique member of the dihydrodipicolinate synthase/N-acetylneuraminate lyase protein family, and its activity was competitively inhibited by pyruvate, a common substrate for other dihydrodipicolinate synthase/N-acetylneuraminate lyase proteins. Furthermore, disruption of Pyr4H2C deaminase genes led to loss of growth on L-hydroxyproline (as well as D-hydroxyproline) but not L-and D-proline, indicating that this pathway is related only to L-hydroxyproline degradation, which is not linked to proline metabolism.