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 (Ϸ5 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. ⌬roxA 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 Ϸ3% 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.

RoxB Is a Novel Type of Rubber Oxygenase That Combines Properties of Rubber Oxygenase RoxA and Latex Clearing Protein (Lcp)

“…The approach to integrate an inducible roxA Xsp gene into a ΔroxA background of Xanthomonas sp. strain 35Y had been previously used successfully for RoxA Xsp expression (11). When we cultivated a clone of the constructed ΔroxA Xanthomonas sp.…”

“…Attempts to express roxB Xsp in Escherichia coli were not successful (not shown). The growth-inhibitory effect of plasmid-derived expression of roxA in E. coli has been described previously (11,28). We therefore integrated roxB Xsp under the control of an L-rhamnose-inducible promoter into the genome of a ΔroxA background of Xanthomonas sp.…”

“…RoxA Xsp was identified as a c-type diheme dioxygenase (7)(8)(9). Recently, the three-dimensional RoxA Xsp structure and essential amino acids of the active site were determined (10,11). RoxA homologs were identified in other Gram-negative bacteria such as Haliangium ochraceum and some myxobacteria (12) but not in any Grampositive rubber degrader.…”

Latex Clearing Protein (Lcp) of Streptomyces sp. Strain K30 Is a b -Type Cytochrome and Differs from Rubber Oxygenase A (RoxA) in Its Biophysical Properties

“…RoxA was purified from a ⌬roxA Xanthomonas sp. with chromosomally integrated roxA plasmid (pNH1::roxA) under rhamnose control as described previously (12,21). Lcp with the replacement of the TAT sequence by a Strep-tag was purified by using E. coli JM109 harboring p4782.1::lcp (volume, 4.8 liters) grown at 22°C for 20 h. The cells were harvested and resuspended in buffer A (1 ml buffer A/g cells, 100 mM potassium phosphate buffer [KPP], pH 7.7, 150 mM sodium chloride).…”

“…strain 35Y (9). Biochemical and biophysical investigation revealed that RoxA is an extracellular dioxygenase with two covalently attached heme groups (10,11) and is structurally but not functionally related to cytochrome c peroxidases (12,13). RoxA cleaves poly(cis-1,4-isoprene) to 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD; C 15 -tri-isoprenoid) as a main end product.…”