Amino acid and sequence analysis of the cytochrome and flavoprotein subunits of p-cresol methylhydroxylase

Abstract

The flavocytochrome p-cresol methylhydroxylase from Pseudomonas putida has been reported to have a Mr of 114,000 and to consist of two subunits, a flavoprotein and a cytochrome c, each with a Mr of 58,000. Recent X-ray crystallographic data from our laboratories [Shamala, N., Lim, L. W., Mathews, F. S., McIntire, W., Singer, T. P., & Hopper, D. J. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 4626-4630], however, indicate an alpha 2 beta 2 structure and a much lower molecular mass (approximately 8000) for the cytochrome subunit. In this paper we report data confirming the conclusions of X-ray crystallographic analysis. From quantitative amino acid analysis, the molecular mass of the flavoprotein monomer is shown to be 48,600 +/- 2200 and that of the cytochrome 8780 +/- 250. These values have been confirmed by gel electrophoresis under denaturing conditions. Gel chromatography under nondenaturing conditions shows that the isolated flavoprotein exists as a dimer, whereas the isolated cytochrome is a monomer. The complete amino acid sequence of the cytochrome c subunit is presented and is shown to have regions of homology to other bacterial c-type cytochromes. The partial N-terminal amino acid sequence (56 amino acids) of the flavoprotein subunit is also reported. The implications of the now established tetrameric structure of the flavocytochrome on data in the literature regarding the redox and association properties of the subunits are examined.

Heterologous Expression in Pseudomonas aeruginosa and Purification of the 9.2-kDa c-Type Cytochrome Subunit of p-Cresol Methylhydroxylase

“…An anomolous behavior during SDS-PAGE analysis has been previously reported for the nonrecombinant PchC subunit of PCMH, giving an apparent relative molecular mass value that is less than that of its true value (21). As shown in Fig.…”

“…This was accomplished by placing the ATG translational start codon of the pchC structural gene encoding the preprotein within the context of a NdeI restriction enzyme site and cloning into the expression vector pET11a. Vectors for the production of PchC in E. coli without its signal peptide sequence were constructed as follows: an ATG translational start codon within the context of a NdeI (CA/TATG) cloning site was introduced immediately preceding the mature N-terminus [Asp34 (8,21)] of the pchC structural gene by using PCR. Plasmid pUC-9869SmaA [pUC18 harboring a 2.9-kb SmaI fragment from P. putida 9869 plasmid pRA500 (2,8) within its SmaI site) served as template with oligonucleotides oPchC5a (5Ј-GCG.GGA.CTC.GAG.CAT.ATG.GAC.AGC.CAG.…”

“…Toward this goal, two pET-based expression vectors were constructed (see Materials and Methods) that, in the first case, placed the mature PchC coding sequence (8,21) immediately following the initiating Met (pETPchC MD ) and, in the second case, the sequence AlaGlnAla that preceeds the mature PchC sequence was retained between the initiating Met and the mature PchC sequence (pET-PchC MAQAD ). This latter arrangement was designed to mimic the N-terminal sequence (MAQAD) of the recombinant cytochrome c 552 overproduced by Keightley et al (13).…”

Identification and molecular characterization of the eugenol hydroxylase genes (ehyA/ehyB) of Pseudomonas sp. strain HR199

“…Since the amino acid sequence deduced from gene ehyA exhibited a typical leader peptide structure (amino acids 1-28), the calculated mass of the ehyA gene product was higher than the molecular masses reported for other α-subunits of flavocytochromes c, e.g., 8,780 for the α-subunit of p-cresol methylhydroxylase from Pseudomonas putida (McIntire et al 1986) or 358 Fig. 3 Alignment of the α-subunit of eugenol hydroxylase from Pseudomonas sp.…”

Recognition of related proteins by iterative template refinement (ITR)

“…Most of these proteins have low sequence similarity with rice cytochrome c and, historically, structural data were necessary to confirm the close relationship among the different class I subgroups (Ambler, 1991). The search also identified the proteinp-cresol methylhydroxylase, which is known to consist of 2 subunits: a flavoprotein and a C-type cytochrome (McIntire et al, 1986). Analysis of the crystal structure of this protein has verified that the cytochrome subunit belongs to the class I family of cytochrome c proteins (Mathews et al, 1991).…”