Characterization of a Unique Pathway for 4-Cresol Catabolism Initiated by Phosphorylation in Corynebacterium glutamicum

Abstract

4-Cresol is not only a significant synthetic intermediate for production of many aromatic chemicals, but also a priority environmental pollutant because of its toxicity to higher organisms. In our previous studies, a gene cluster implicated to be involved in 4-cresol catabolism, creCDEFGHIR, was identified in Corynebacterium glutamicum and partially characterized in vivo. In this work, we report on the discovery of a novel 4-cresol biodegradation pathway that employs phosphorylated intermediates. This unique pathway initiates with the phosphorylation of the hydroxyl group of 4-cresol, which is catalyzed by a novel 4-methylbenzyl phosphate synthase, CreHI. Next, a unique class I P450 system, CreJEF, specifically recognizes phosphorylated intermediates and successively oxidizes the aromatic methyl group into carboxylic acid functionality via alcohol and aldehyde intermediates. Moreover, CreD (phosphohydrolase), CreC (alcohol dehydrogenase), and CreG (aldehyde dehydrogenase) were also found to be required for efficient oxidative transformations in this pathway. Steady-state kinetic parameters (K m and k cat ) for each catabolic step were determined, and these results suggest that kinetic controls serve a key role in directing the metabolic flux to the most energy effective route.

Elucidation of gibberellin biosynthesis in bacteria reveals convergent evolution

“…Thus it seems that while CYP114 is catalytically capable of the full transformation of 4 to 7 , SDR GA is necessary for the efficient oxidation of 6 to 7 to occur. The use of a dehydrogenase for this type of chemical transformation is not uncommon, as it has been shown in biosynthetic pathways for other metabolites that the relevant CYPs do not efficiently catalyze full oxidation to the carboxylate, leading to a requirement for alcohol and/or aldehyde dehydrogenases for complete conversion 30–32 . However, neither plant nor fungal GA biosynthesis rely on a dedicated dehydrogenase 1 , and therefore this enzyme is unique to the bacterial pathway.…”

Selective oxidation of aliphatic C–H bonds in alkylphenols by a chemomimetic biocatalytic system

“…The P450 enzyme CreJ (also named CYP288A2 by Nelson et al) (31) catalyzes the successive oxidations from 1′→1′a→1′b→1′c in the presence of redox partners CreE and CreF, and the electron donor NADPH (30). Although different oxidative modifications of the distal methyl group are well tolerated by CreJ, the tethered phosphate group is required for the activity of this P450 enzyme because the nonphosphorylated substrate cannot be recognized (30). To understand this unique substrate recognition mechanism, the X-ray crystal structure of CreJ in complex with 1′ was solved at 2.0 Å (PDB ID code: 5GWE).…”

“…2A) in the Gram-positive bacterium Corynebacterium glutamicum (30). Initially, 1 is phosphorylated to 1′ by a novel 4-methylphenyl phosphate synthase, CreHI, with consumption of ATP.…”

Cyclohexane, naphthalene, and diesel fuel increase oxidative stress, CYP153, sodA, and recA gene expression in Rhodococcus erythropolis

“…Such enzymes were discovered also in Rhodococcus. Other studies have shown that cytochromes can oxidize both linear alkanes and aromatic hydrocarbons (Bell & Wong, 2007;Du et al, 2006;Ignatovets, Akhramovich, & Leontiev, 2009).…”