Uncoupling of the cytochrome P-450cam monooxygenase reaction by a single mutation, threonine-252 to alanine or valine: possible role of the hydroxy amino acid in oxygen activation.

Abstract

Site-directed mutants of cytochrome P-450cam (the cytochrome P-450 that acts as the terminal monooxygenase in the d-camphor monooxygenase system), in which threonine-252 had been changed to alanine, valine, or serine, were employed to study the role of the hydroxy amino acid in the monooxygenase reaction. The mutant enzymes were expressed in Escherichia coli and were purified by a conventional method. All the mutant enzymes in the presence of d-camphor exhibited optical absorption spectra almost indistinguishable from those of the wild-type enzyme in their ferric, ferrous, oxygenated, and carbon monoxide ferrous forms. In a reconstituted system with putidaredoxin and its reductase, the alanine enzyme consumed O2 at a rate (1100 per min per heme) comparable to that of the wild-type enzyme (1330 per min per heme), whereas the amount of exo-5-hydroxycamphor formed was less than 10% of that formed by the wild-type enzyme. About 85% of the O2 consumed was recovered as H2O2. The valine enzyme also exhibited an oxidase activity to yield H2O2 accompanied by a relative decrease in the monooxygenase activity. On the other hand, the serine enzyme exhibited essentially the same monooxygenase activity as that of the wild-type enzyme. Thus, uncoupling of O2 consumption from the monooxygenase function was produced by the substitution of an amino acid without a hydroxyl group. When binding of O2 to the ferrous forms was examined, the alanine and valine enzymes formed instantaneously an oxygenated form, which slowly decomposed to the ferric form with rates of 5.5 and 3.2 x 10(-3) sec-1 for the former and latter enzymes, respectively. Since these rates were too slow to account for the overall rates of O2 consumption, the formation of H2O2 was considered to proceed not by way of this route but through the decomposition of a peroxide complex formed by reduction of the oxygenated form by reduced putidaredoxin. Based on these findings, a possible mechanism for oxygen activation in this monooxygenase reaction has been discussed.

Efficient catalytic turnover of cytochrome P450cam is supported by a T252N mutation

“…The T252N and T252N/V253T mutants, like the wild-type protein, showed strong camphor hydroxylation activities. However, as previously reported [6], the camphor hydroxylation activity of the T252A mutant was negligible (Fig. 4).…”

“…The crystal structures of ferrous camphor-and CO-bound P450 cam suggest that Thr252 is a key catalytic residue. This inference is substantiated by the fact that mutation of Thr252 to an Ala, Val, or Leu produces enzymes in which substrate hydroxylation is highly uncoupled from electron transfer [4][5][6][7], whereas replacement by a Ser yields a catalytically active enzyme [6]. In one instance is the conserved Thr replaced by a non-hydrogen bonding moiety.…”

Determinants governing the CYP74 catalysis: Conversion of allene oxide synthase into hydroperoxide lyase by site‐directed mutagenesis

“…The previously described P450 mutant forms [19][20][21][22][23][24], including the MtHPL mutant [18], possessed some alterations like the change of substrate specificity or regiospecificity, but not the qualitative transformations of catalysis (like the conversion of dehydrase (AOS) into isomerase (HPL) in the present work). ysis, either loses one hydrogen atom, affording the allene oxide (WT enzyme), or undergoes the rearrangement into the isomeric radical, which recombines with hydroxyl radical to form the hemiacetal, a primary HPL product (F295I and S297A mutant forms).…”

Molecular cloning and heterologous expression of a cDNA encoding berbamunine synthase, a C--O phenol-coupling cytochrome P450 from the higher plant Berberis stolonifera.

“…With the exception of allene oxide synthase (13), the cytochrome P450s for which cDNAs have been isolated are monooxygenases. Multiple structure-function studies have been performed on the by far best-studied cytochrome P450, the bacterial cytochrome P450cam, which suggests that three amino acid residues of the distal helix (helix I), Gly-248, Gly-249, and Thr-252 (Table 2), are essential for formation of the oxygen-binding pocket (28,31). Mutation of Thr-252 to alanine or valine resulted in uncoupling of the monooxygenase reaction (31).…”

“…Multiple structure-function studies have been performed on the by far best-studied cytochrome P450, the bacterial cytochrome P450cam, which suggests that three amino acid residues of the distal helix (helix I), Gly-248, Gly-249, and Thr-252 (Table 2), are essential for formation of the oxygen-binding pocket (28,31). Mutation of Thr-252 to alanine or valine resulted in uncoupling of the monooxygenase reaction (31). Interestingly, Thr-252 is present in berbamunine synthase, although there is no monooxygenation reaction in the C-O phenol-coupling process.…”