Crystal structure of a ferredoxin reductase for the CYP199A2 system from Rhodopseudomonas palustris

Abstract

Cytochrome P450-199A2 from Rhodopseudomonas palustris oxidizes para-substituted benzoic acids and may play a role in lignin and aromatic acid degradation pathways in the bacterium. CYP199A2 has an associated [2Fe-2S] ferredoxin, palustrisredoxin (Pux) but not a ferredoxin reductase. A genome search identified the palustrisredoxin reductase (PuR) gene. PuR was produced in Escherichia coli and shown to be a flavin-dependent protein that supports efficient electron transfer from NADH to Pux, thus reconstituting CYP199A2 monooxygenase activity (k(cat) = 37.9 s(-1) with 4-methoxybenzoic acid). The reduction of Pux by PuR shows K(m) = 4.2 microM and k(cat) = 262 s(-1) in 50 mM Tris, pH 7.4. K(m) is increased to 154 microM in the presence of 200 mM KCl, indicating the importance of ionic interactions in PuR/Pux binding. The crystal structure of PuR has been determined at 2.2 A resolution and found to be closely related to that of other oxygenase-coupled NADH-dependent ferredoxin reductases. Residues on the surface that had been proposed to be involved in ferredoxin reductase-ferredoxin binding are conserved in PuR. However, Lys328 in PuR lies over the FAD isoalloxazine ring and, together with His11 and Gln41, render the electrostatic potential of the surface more positive and may account for the greater involvement of electrostatic interactions in ferredoxin binding by PuR. Consistent with these observations the K328G mutation weakened Pux binding and virtually eliminated the dependence of PuR/Pux binding on salt concentration, thus confirming that the FAD si side surface in the vicinity of Lys328 is the ferredoxin binding site.

Molecular Characterization of a Class I P450 Electron Transfer System from Novosphingobium aromaticivorans DSM12444

“…Putidaredoxin (Pdx) is well known to have an effector role in CYP101A1 activity (12,13). The activity of CYP199A2 from Rhodopseudomonas palustris CGA009 has been reconstituted with palustrisredoxin (Pux), a [2Fe-2S] ferredoxin genomically associated with CYP199A2, and an ONFR, palustrisredoxin reductase (PuR) (14). The high demethylation activity of this system is severely compromised in the hybrid PdR/Pdx/CYP199A2 system (14) due to weak ferredoxin/P450 binding (14,15).…”

“…The activity of CYP199A2 from Rhodopseudomonas palustris CGA009 has been reconstituted with palustrisredoxin (Pux), a [2Fe-2S] ferredoxin genomically associated with CYP199A2, and an ONFR, palustrisredoxin reductase (PuR) (14). The high demethylation activity of this system is severely compromised in the hybrid PdR/Pdx/CYP199A2 system (14) due to weak ferredoxin/P450 binding (14,15). Numerous P450 enzymes with potentially interesting and desirable activities are orphaned in the genome; the electron transfer proteins are not located nearby in the gene sequence.…”

The importance of the benzoic acid carboxylate moiety for substrate recognition by CYP199A4 from Rhodopseudomonas palustris HaA2

“…[1] The closely related CYP199A2 enzyme, from the CGA009 strain of R. palustris, has been reported to be a biocatalyst for the oxyfunctionalisation of 4-methoxybenzoic acid and various other aromatic carboxylic acids including 2-naphthoic, indole-6-carboxylic and cinnamic acids. [7,[17][18][19][20] Recently, the wild-type enzyme and mutant forms of CYP199A2…”

“…For example the Ser247 residue of CYP199A2, which interacts with the carboxylate group of 4-methoxybenzoic acid (Ser247 is equivalent to Ser244 in CYP199A4) has been mutated to an aspartate to improve the activity of this enzyme for the oxidation of phenols. [21] A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 20 …”

Structural and thermodynamic characterization of the adrenodoxin-like domain of the electron-transfer protein Etp1 from Schizosaccharomyces pombe

“…So far, no crystal structure of an eukaryotic adrenodoxin-type ferredoxin involved in heme A synthesis and assembly of iron-sulfur clusters has been described, whereas three structures of bacterial proteins with corresponding function are already solved [13][14][15][16]. The corresponding E. coli ferredoxin gene was shown to be crucial for the efficient biosynthesis of Fe-S clusters in vivo [17,18], and the encoded protein was proven to support in vitro assembly of Fe-S clusters [19].…”