Identification of a self‐sufficient cytochrome P450 monooxygenase from Cupriavidus pinatubonensis JMP134 involved in 2‐hydroxyphenylacetic acid catabolism, via homogentisate pathway

Abstract

The self-sufficient cytochrome P450 RhF and its homologues belonging to the CYP116B subfamily have attracted considerable attention due to the potential for biotechnological applications based in their ability to catalyse an array of challenging oxidative reactions without requiring additional protein partners. In this work, we showed for the first time that a CYP116B self-sufficient cytochrome P450 encoded by the ohpA gene harboured by Cupriavidus pinatubonensis JMP134, a b-proteobacterium model for biodegradative pathways, catalyses the conversion of 2-hydroxyphenylacetic acid (2-HPA) into homogentisate. Mutational analysis and HPLC metabolite detection in strain JMP134 showed that 2-HPA is degraded through the well-known homogentisate pathway requiring a 2-HPA 5-hydroxylase activity provided by OhpA, which was additionally supported by heterologous expression and enzyme assays. The ohpA gene belongs to an operon including also ohpT, coding for a substrate-binding subunit of a putative transporter, whose expression is driven by an inducible promoter responsive to 2-HPA in presence of a predicted OhpR transcriptional regulator. OhpA homologues can be found in several genera belonging to Actinobacteria and a-, band c-proteobacteria lineages indicating a widespread distribution of 2-HPA catabolism via homogentisate route. These results provide first time evidence for the natural function of members of the CYP116B selfsufficient oxygenases and represent a significant input to support novel kinetic and structural studies to develop cytochrome P450-based biocatalytic processes.

Enzymatic Control over Reactive Intermediates Enables Direct Oxidation of Alkenes to Carbonyls by a P450 Iron-Oxo Species

“…The physiological substrate of P450 LA1 is unknown, and this enzyme has been shown to catalyze various reactions such as asymmetric sulfoxidation, sp 3 C–H bond hydroxylation, and alkene epoxidation on diverse compounds. A homolog of P450 LA1 within the CYP116B subfamily was found to catalyze aromatic C–H bond hydroxylation in 2-hydroxyphenylacetic acid catabolism, which is the first demonstration of a physiological substrate for an enzyme from the CYP116B subfamily . The evolved aMOx enzyme is the first catalyst to use metal-oxo species for selective alkene to carbonyl oxidation and thus a good opportunity to study the origin of chemoselectivity in this reaction.…”

“…A homolog of P450 LA1 within the CYP116B subfamily was found to catalyze aromatic C−H bond hydroxylation in 2-hydroxyphenylacetic acid catabolism, which is the first demonstration of a physiological substrate for an enzyme from the CYP116B subfamily. 28 The evolved aMOx enzyme is the first catalyst to use metal-oxo species for selective alkene to carbonyl oxidation and thus a good opportunity to study the origin of chemoselectivity in this reaction. As we will describe below, iron-oxo-mediated alkene oxidation can involve a short-lived radical intermediate that is generated with a large excess of the kinetic energy (Figure 1).…”

Identification of a Phylogenetically Divergent Vanillate O-Demethylase from Rhodococcus ruber R1 Supporting Growth on Meta-Methoxylated Aromatic Acids

“…In order to gain comprehension about the whole function of the divergent VanOD from R. ruber R1, a plasmid construct containing the vanAB genes of this strain was introduced into C. pinatubonensis JMP134, a well-known aromatics-degrader bacterium unable to grow on VA, 3-MB, and 5-MS, but that harbors PCA, 3-HB, and gentisate degradation routes ( Figure 2 B,D,F) [ 40 ], allowing complementation of the catabolic abilities. The expression of the vanAB genes was controlled by the L-arabinose-inducible P BAD promoter, which was chosen since L-arabinose is non-toxic and is not a carbon source for C. pinatubonensis JMP134, permitting reliable growth tests in this strain [ 30 , 41 ]. Remarkably, the presence of vanAB genes was sufficient to allow L-arabinose-depending growth on VA, 3-MB, and 5-MS of JMP134 strain ( Figure 2 A,C,E), strongly suggesting that VanOD of R1 strain has O-demethylation activity toward the three meta -methoxylated aromatic substrates.…”

“…In brief, PCR product comprising vanAB genes (locus tags: E2561_01225–E2561_01230), was obtained using oligos FW_vanAB_R1_EcoRI (5′-TGAC GAATTC GAAGGAACGACATGACCGATC-3′) and RV_vanAB_R1_XbaI (5′-GTAC TCTAGA TGTATCCGATGACCAGGCC-3′) including underlined restriction sites for EcoRI and XbaI enzymes. The amplified DNA fragment was purified and double digested to be ligated into EcoRI/XbaI restriction sites of pBS1 [ 30 ], forming pBS1- vanAB plasmid, that was electroporated into E. coli Mach1. Transformed cells were selected in LB medium supplemented with gentamycin 30 µg mL −1 ; and selected transformants were checked by PCR for proper insertion of the vanAB genes.…”

Cupriavidus metallidurans CH34 Possesses Aromatic Catabolic Versatility and Degrades Benzene in the Presence of Mercury and Cadmium

“…The absence of degradative canonical pathways of phenylpropanoids (cinnamate, phenylpropionates) and 4-HPA in Cupriavidus strains B-8, NH9, and JMP134, despite their growth on these compounds [27,31,67], indicate that members of the Cupriavidus genus may degrade them via novel catabolic pathways. Cytochrome P450 OhpA is a self-sufficient cytochrome involved in the transformation of 2-HPA into homogentisate, which was described recently in C. pinatubonensis JMP134 [73], being also present in the genome of strain CH34 (RMET_RS25305), evidencing the need for further characterization of novel enzymatic activities in the Cupriavidus genus.…”