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

Abstract

Cytochrome P450 (CYP) 4 enzymes constitute a superfamily of heme-containing monooxygenases (1, 2) that participate in a variety of biological processes such as carbon source assimilation, biosynthesis and biodegradation, xenobiotic detoxification, and metabolism of medicines (1, 2). The most common activity of P450 enzymes is the insertion of an oxygen atom from dioxygen into chemically inert carbon-hydrogen bonds, but other reaction types including dealkylation, desaturation, heteroatom oxidation, epoxidation, phenol coupling, and reductive dehalogenation are also known (3-6). The various activities of P450 enzymes are of great interest due to their potential applications in, for example, synthesis of fine chemicals and drug metabolites under mild conditions with high specificity.P450 enzymatic activity requires two electrons that are usually derived from NAD(P)H and delivered to the P450s by electron transfer proteins which are broadly divided into two classes (7,8). Class I systems are diverse, usually consisting of an oxygenase-coupled NAD(P)H-dependent ferredoxin reductase (ONFR) and an iron-sulfur ferredoxin. Such systems are the predominant forms in prokaryotes but are also found in eukaryotic mitochondrial membranes. ONFRs typically contain an FAD cofactor. Ferredoxin cluster types include [2Fe-2S], [3Fe-4S], [4Fe-4S], and combinations of these (7, 9). Non-ferredoxin FMN proteins have also been identified (10). Class II P450 enzymes are most common in eukaryotes and utilize an NADPH-cytochrome P450 reductase (CPR) containing prosthetic groups FAD and FMN (11). Recently other more diverse electron transfer systems for P450 enzymes have been discovered and these have been defined into several new classes (7,8).An important difference between the two main classes is that, whereas a single CPR supports the activity of all 57 human P450s and yeast CPRs support the activity of numerous P450s heterologously expressed in the organism, most class I systems show redox partner specificity. 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). Numerous P450 enzymes with potentially interesting and desirable activities are

Conformational selectivity in cytochrome P450 redox partner interactions

“…We also included CYP101D1 (PDB ID code 4C9K) (17) as a control. CYP101D1 is a very close homolog to P450cam and uses camphor as a substrate to give the same product as P450cam (16). Comparing CYP101D1 with P450cam provided insights on the range of motion that the substrate can experience, yet retain regioselective and stereoselective hydroxylation.…”

“…We also carried out a control simulation with another P450, CYP101D1. CYP101D1 is a very close homolog to P450cam, with an rms deviation of 1.0 Å for backbone atoms and has a very similar active site structure (16). CYP101D1 catalyzes exactly the same reaction as P450cam at the same rate giving the same single product (16).…”

“…CYP101D1 is a very close homolog to P450cam, with an rms deviation of 1.0 Å for backbone atoms and has a very similar active site structure (16). CYP101D1 catalyzes exactly the same reaction as P450cam at the same rate giving the same single product (16). However, CYP101D1 is more promiscuous, with respect to redox partner selectivity because Pdx can support CYP101D1 catalysis despite not being its natural redox partner (17).…”

Chain length-dependent cooperativity in fatty acid binding and oxidation by cytochrome P450BM3 (CYP102A1)

“…The first electron transfer is rate limiting for a number of bacterial P450 systems including P450 cam (CYP101A1) from Pseudomonas putida (Brewer and Peterson, 1988), CYP199A2 and CYP199A4 from Rhodopseudomonas palustris (Bell et al, 2010b(Bell et al, , 2010c) and CYP101D1 from Novosphingobium aromaticivorans Bell et al, 2010a;Yang et al, 2010). It has been shown that the first flavin-to-heme electron transfer, though one of the slower steps in the overall catalytic cycle, is not rate limiting for palmitate and arachidonate oxidation by P450 BM3 (Munro et al, 1996;Ost et al, 2003;Whitehouse et al, 2011).…”

Biodegradation and Bioremediation of Organic Pesticides

“…They can catalyze reactions of oxidation, reduction or oxidative breakdown of xenobiotics ( Figure 2). It seems that they are evolutively conserved since genomes from virus, bacteria, algae, plant, fungi and animals have isoforms of CYP codified (12)(13)(14)(15)(16)(17)(18)(19)(20)(21). In eukaryotic organisms, CYP is found in smooth endoplasmic reticulum, and can biotransform a wide range of pollutants.…”