(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) > cellular organisms: NE > Bacteria: NE > Proteobacteria: NE > Gammaproteobacteria: NE > Pseudomonadales: NE > Pseudomonadaceae: NE > Pseudomonas: NE > Pseudomonas sp.: NE
LegendThis sequence has been compared to family alignement (MSA) red => minority aminoacid blue => majority aminoacid color intensity => conservation rate title => sequence position(MSA position)aminoacid rate Catalytic site Catalytic site in the MSA MFKPVVKSRSSRSFCYLAGCLAMVAATLSSTAQAKSEWACPEGFTPKAGL NTDFPSDGKKRAFVVVPPKDSAGGAPVWVPMVGTVEATNWNLNVPRSGNN AKLAEHGYMVISPVRQCAEQDPNLGAGACNGVGKDGWTWNPWNDGRAPDA SGDKYKTDAGDDVRFLEAMVRCVGTKWKLDRKRLFLGGISAGGTMTNRAL LFDSEFWAGGMPISGEWYSTKDDGSTVPFQETRKMVAAAPAKIWQGRVGP YPLPSKLDPMVVITVWGGEKDLWDCGPPLGLCSDYRPTTQASSNYFSSIS NVVHVACSATHGHMWPQVNTDAFNLWALNTMASHPKGSSPKDFKLTAPPE GYSCKIGRFTDHYK
The ever-increasing production and use of polyvinyl alcohol (PVA) threaten our environment. Yet PVA can be assimilated by microbes in two steps: oxidation and cleavage. Here we report novel alpha/beta-hydrolase structures of oxidized PVA hydrolase (OPH) from two known PVA-degrading organisms, Sphingopyxis sp. 113P3 and Pseudomonas sp. VM15C, including complexes with substrate analogues, acetylacetone and caprylate. The active site is covered by a lid-like beta-ribbon. Unlike other esterase and amidase, OPH is unique in cleaving the CC bond of beta-diketone, although it has a catalytic triad similar to that of most alpha/beta-hydrolases. Analysis of the crystal structures suggests a double-oxyanion-hole mechanism, previously only found in thiolase cleaving beta-ketoacyl-CoA. Three mutations in the lid region showed enhanced activity, with potential in industrial applications.
Title: Roles of tryptophan residue and disulfide bond in the variable lid region of oxidized polyvinyl alcohol hydrolase Yang Y, Ko TP, Liu L, Li J, Huang CH, Chen J, Guo RT, Du G Ref: Biochemical & Biophysical Research Communications, 452:509, 2014 : PubMed
Oxidized polyvinyl alcohol hydrolase (OPH) catalyzes the cleavage of C-C bond in beta-diketone. It belongs to the alpha/beta-hydrolase family and contains a unique lid region that covers the active site. The lid is the most variable region when pOPH from Pseudomonas sp. VM15C and sOPH from Sphingopyxis sp. 113P3 are compared. The wild-type enzymes and the pOPH mutants W255A, W255Y and W255F were analyzed for lipase activity by using p-nitrophenyl (pNP) esters as the substrates. The wild-type enzymes showed increased Km and decreased kcat/Km with the acyl chain length, and the mutants showed reduced kcat/Km for pNP acetate, indicating the importance of Trp255 in sequestering the active site from solvent. The significantly lower activity for pNP butyrate can be a result of product inhibition, as suggested by the complex crystal structures, in which butyric acid, DMSO or PEG occupied the same substrate-binding cleft. The mutant activity was retained with pNP caprylate and pNP laurate as the substrates, reflecting the amphipathic nature of the cleft. Moreover, the disulfide bond formation of Cys257/267 is important for the activity of pOPH, but it is not essential for sOPH, which has a shorter lid structure.
Sphingopyxis sp. (strain 113P3) Oxidized polyvinyl alcohol hydrolase
