Properties of p-Cresol Methylhydroxylase Flavoprotein Overproduced by Escherichia coli

Abstract

The alpha(2)beta(2) flavocytochrome p-cresol methylhydroxylase (PCMH) from Pseudomonas putida is composed of a flavoprotein homodimer (alpha(2) or PchF(2); M(r) = 119 kDa) with a cytochrome monomer (beta, PchC; M(r) = 9.3 kDa) bound to each PchF subunit. Escherichia coli BL21(DE3) has been transformed with a vector for expression of the pchF gene, and PchF is overproduced by this strain as the homodimer. During purification, it was recognized that some PchF had FAD bound, while the remainder was FAD-free. However, unlike PchF obtained from PCMH purified from P. putida, FAD was bound noncovalently. The FAD was conveniently removed from purified E. coli-expressed PchF by hydroxyapatite chromatography. Fluorescence quenching titration indicated that the affinity of apo-PchF for FAD was sufficiently high to prevent the determination of the dissociation constant. It was found that p-cresol was virtually incapable of reducing PchF with noncovalently bound FAD (PchF(NC)), whereas 4-hydroxybenzyl alcohol, the intermediate product of p-cresol oxidation by PCMH, reduced PchF(NC) fairly quickly. In contrast, p-cresol rapidly reduced PchF with covalently bound FAD (PchF(C)), but, unlike intact PCMH, which consumed 4 electron equiv/mol when titrated with p-cresol (2 electrons from p-cresol and 2 from 4-hydroxybenzyl alcohol), PchF(C) accepted only 2 electron equiv/mol. This is explained by extremely slow release of 4-hydroxybenzyl alcohol from reduced PchF(C). 4-Hydroxybenzyl alcohol rapidly reduced PchF(C), producing 4-hydroxybenzaldehyde. It was demonstrated that p-cresol has a charge-transfer interaction with FAD when bound to oxidized PchF(NC), whereas 4-bromophenol (a substrate analogue) and 4-hydroxybenzaldehyde have charge-transfer interactions with FAD when bound to either PchF(C) or PchF(NC). This is the first example of a "wild-type" flavoprotein, which normally has covalently bound flavin, to bind flavin noncovalently in a stable, redox-active manner.

Biosynthesis of Covalently Bound Flavin: Isolation and in Vitro Flavinylation of the Monomeric Sarcosine Oxidase Apoprotein

“…However, the plot does not intersect the origin, as would be expected for a second order reaction, suggesting that covalent attachment may involve prior formation of an unstable noncovalent E·FAD complex. In contrast, the flavin-binding subunit from p-cresol methylhydroxylase exhibits only noncovalent binding with FAD (34). Covalent attachment of FAD is triggered by a chaperone-like action of the heme-binding subunit that induces a conformational change in the flavin-binding subunit (13).…”

Cloning, Sequencing, and Heterologous Expression of the Murine Peroxisomal Flavoprotein, N1-Acetylated Polyamine Oxidase

“…The anaerobic mPAO solution contained also 50 mM D-glucose, 3 g of catalase, and 50 g of glucose oxidase to scavenge trace dissolved O 2 . The spectral data were subjected to "Factor Analysis" using the SPECFIT program (Spectrum Software Associates, Chapel Hill, NC) as described earlier (40,41). A 1.20 M solution of mPAO was titrated anaerobically with a solution of 0.5 mM N 1 -acetyl-SPD in 50 mM KH 2 PO 4 /KOH buffer, pH 7.6, at 25°C.…”

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

“…Recently, this laboratory constructed an expression vector, pET-PchC, for the production of recombinant PchC in E. coli (10). 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.…”

“…Toward these goals, the PchF subunit has been overproduced independent of the cytochrome subunit in Escherichia coli, and about 25 mg may be obtained in purified form from 6 liters of cell culture (10). The recombinant flavoprotein is obtained in its dimeric state with FAD noncovalently bound, which may be removed by hydroxyapatite chromatography (10).…”

“…The recombinant flavoprotein is obtained in its dimeric state with FAD noncovalently bound, which may be removed by hydroxyapatite chromatography (10). The production of the PchC cytochrome subunit in recombinant form has proven, however, to be problematic.…”