Resolution of the flavocytochrome p-cresol methylhydroxylase into subunits and reconstitution of the enzyme

Abstract

An improved procedure is described for the isolation of the flavocytochrome p-cresol methylhydroxylase (PCMH) from Pseudomonas putida as well as methods for the separation of its subunits in native form and their recombination to reconstitute the original flavocytochrome. Under appropriate conditions, the reconstitution is stoichiometric and results in complete recovery of the catalytic activity of the flavocytochrome. The separated flavoprotein subunit shows only 2% of the catalytic activity of the original enzyme on p-cresol and is characterized by converging lines in bisubstrate kinetic analysis, while the intact and reconstituted enzymes show parallel line kinetics in steady-state experiments. van't Hoff plots of the dependence of the dissociation constant of the subunits of PCMH on temperature show a break near 15 degrees C. Above this temperature, KD is characterized by a positive delta H value of 12.6 kcal mol-1; below 15 degrees C, the dissociation is essentially temperature independent. The subunit dissociation is strongly dependent on ionic strength in the oxidized form of PCMH but not in the reduced form of the enzyme. Reduction also lowers the KD significantly, while substrates and nonoxidizable competitive inhibitors lower the dissociation constant even further, suggesting a conformation change. Combination of the subunits to form PCMH entails a small but measurable change in the absorption spectra of the component proteins.

Cloning, sequencing, and expression of the structural genes for the cytochrome and flavoprotein subunits of p-cresol methylhydroxylase from two strains of Pseudomonas putida

“…The resulting molar ratios are given in parentheses. Since a2 PchF has about 2% of the specific activity of a212 PCMH (35,36), as the level of PchC was increase, progressively less PchF was used.…”

“…Its 3-A (1 A = 0.1 nm) three-dimensional structure is known (42) stereochemistry of its oxidation ofp-cresol, 4-ethylphenol, and 1-(4'-hydroxyphenyl)ethanol (45,46). The flavoprotein and cytochrome subunits have been separated, characterized, and reconstituted into native enzyme (35,36). Further, the amino acid sequences of the entire cytochrome subunit and the N terminus and flavin attachment site of the flavoprotein of PCMH69A have been determined (44,48).…”

“…The flavoprotein and cytochrome subunits have been separated, characterized, and reconstituted into native enzyme (35,36). Further, the amino acid sequences of the entire cytochrome subunit and the N terminus and flavin attachment site of the flavoprotein of PCMH69A have been determined (44,48).…”

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

“…Each of the latter subunits has flavin adenine dinucleotide (FAD) covalently attached via a 8␣-O-tyrosyl-FAD linkage to Tyr384 (1,2). PCMH has been extensively studied in this laboratory, including its isolation (3), determination of the steady-state kinetic mechanism (4,5), and the resolution of the tetramer into its subunits by isoelectric focusing (3,6). The reassembly of the holoenzyme, including the selfcatalytic covalent attachment of the FAD, is achieved simply by mixing apoPchF and PchC in the presence of FAD (7).…”

“…Although small amounts have been produced and isolated from E. coli JM109 and DH5␣ harboring a pKK223-3 vector derivative (2,7), a supply of the cytochrome has generally been obtained by dismantling the holoenzyme isolated from P. putida NCIMB 9869 by isoelectric focusing (3,6).…”