title: Identification and characterization of D-hydroxyproline dehydrogenase and Delta1-pyrroline-4-hydroxy-2-carboxylate deaminase involved in novel L-hydroxyproline metabolism of bacteria: metabolic convergent evolution.
abstract: 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 α-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 Δ(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 (α(4)β(4)γ(4)), and two FADs, FMN, and [2Fe-2S] iron-sulfur cluster were contained in αβγ 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.
authors: Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan. irab@agr.ehime-u.ac.jp
authors: Seiya
authors: S
authors: Watanabe
authors: Daichi
authors: D
authors: Morimoto
authors: Fumiyasu
authors: F
authors: Fukumori
authors: Hiroto
authors: H
authors: Shinomiya
authors: Hisashi
authors: H
authors: Nishiwaki
authors: Miyuki
authors: M
authors: Kawano-Kawada
authors: Yuuki
authors: Y
authors: Sasai
authors: Yuzuru
authors: Y
authors: Tozawa
authors: Yasuo
authors: Y
authors: Watanabe
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019759:Carbon-Nitrogen Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019143:Evolution, Molecular; D006909:Hydroxyproline / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D010088:Oxidoreductases; D019759:Carbon-Nitrogen Lyases; D006909:Hydroxyproline