title: Phe317 is essential for rubber oxygenase RoxA activity.
abstract: RoxA  is an extracellular c-type diheme cytochrome secreted by Xanthomonas sp. strain 35Y during growth on rubber. RoxA cleaves poly(cis-1,4-isoprene) to 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD). Analysis of the RoxA structure revealed that Phe317 is located in close proximity (≈5 Å) to the N-terminal heme that presumably represents the active site. To find evidence of whether Phe317 is important for catalysis, we changed it to tyrosine, tryptophan, leucine, histidine, or alanine. All five RoxA muteins were expressed after integration of the respective gene into the chromosome of a Xanthomonas sp. ΔroxA strain. Residual clearing zone formation on opaque latex agar was found for Xanthomonas sp. strains expressing the Phe317Leu, Phe317Ala, or Phe317His variant (wild type > Leu > Ala > His). Strains in which Phe317 was changed to tyrosine or tryptophan were inactive. Phe317Ala and Phe312Leu RoxA muteins were purified, and polyisoprene cleavage activities were reduced to ≈3% and 10%, respectively. UV-visible spectroscopy of RoxA muteins confirmed that both heme groups were present in an oxidized form, but spectral responses to the addition of low-molecular-weight (inhibitory) ligand molecules such as imidazole and pyridine were different from those of wild-type RoxA. Our results show that residue 317 is involved in interaction with substrates. This is the first report on structure-function analysis of a polyisoprene-cleaving enzyme and on the identification of an amino acid that is essential for polyisoprene cleavage activity.
authors.affiliation: Institut für Mikrobiologie, Universität Stuttgart, Stuttgart, Germany.
authors.forename: Jakob
authors.initials: J
authors.lastname: Birke
authors.forename: Nadja
authors.initials: N
authors.lastname: Hambsch
authors.forename: Georg
authors.initials: G
authors.lastname: Schmitt
authors.forename: Josef
authors.initials: J
authors.lastname: Altenbuchner
authors.forename: Dieter
authors.initials: D
authors.lastname: Jendrossek
mesh_terms: D019943:Amino Acid Substitution; D020134:Catalytic Domain; D006418:Heme / Q000032:analysis; D007840:Latex / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010649:Phenylalanine / Q000235:genetics* / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D014974:Xanthomonas / Q000201:enzymology* / Q000235:genetics
chemical_list: D007840:Latex; D050505:Mutant Proteins; D006418:Heme; D010649:Phenylalanine; D010105:Oxygenases