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 (Km and kcat) 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.

Keywords: 4-cresol; Corynebacterium glutamicum; biodegradation; cytochrome P450; gram-positive bacteria; microbiology; phosphorylation.

FIGURE 1.

Known pathways for 4-cresol catabolism by variant microorganisms. Degradation of 4-cresol begins from methyl hydroxylation (A), linking fumarate to the methyl group (B), and direct aromatic ring hydroxylation (C).

FIGURE 2.

CreHI catalyzed reactions. A, upper panel, CreHI reaction scheme. Lower panel, HPLC analysis (275 nm) of CreHI catalyzed reactions. s1 and s2, authentic standards; i, 4-cresol as substrate; ii, 4-hydroxybenzyl alcohol as substrate; iii, 4-hydroxybenzyl aldehyde as substrate; iv, 4-hydroxybenzoate as substrate. Traces in blue represent corresponding control reactions without the addition of enzymes. B, HRMS of 4-methylbenzyl phosphate. C, HRMS of benzylalcohol-4-phosphate. D, HRMS of benzylaldehyde-4-phosphate. Note that because of distinct extinction coefficients, the peak intensity of compounds do not necessarily reflect relative amounts.

FIGURE 3.

CreJEF catalyzed reactions. A, upper panel, CreJEF reactions scheme. Lower panel, HPLC analysis (275 nm) of CreJEF catalyzed reactions. s1 and s2, authentic standards; i, 4-methylbenzyl phosphate as substrate; ii, benzylalcohol-4-phosphate as substrate; iii, benzylaldehyde-4-phosphate as substrate. Traces in blue represent corresponding control reactions without addition of enzymes. B, HRMS of benzoate-4-phosphate. Note that because of distinct extinction coefficients, the peak intensity of compounds do not necessarily reflect relative amounts.

FIGURE 4.

Reactions catalyzed by CreG and CreC. A, upper panel, CreG reactions scheme. Lower panel, HPLC analysis (275 nm) of CreG catalyzed reactions. s1 and s2, authentic standards; i, 4-hydroxybenzyl alcohol as substrate; ii, benzylalcohol-4-phosphate as substrate. B, upper panel, CreC reactions scheme. Lower panel, HPLC analysis of CreC catalyzed reactions. s1 and s2, authentic standards; i, 4-hydroxybenzyl aldehyde as substrate; ii, benzylaldehyde-4-phosphate as substrate. Traces in blue represent corresponding control reactions without addition of enzymes. Note that because of distinct extinction coefficients, the peak intensity of compounds do not necessarily reflect relative amounts.

FIGURE 5.

Reactions catalyzed by CreD. Upper panel, CreD reactions scheme. Lower panel, HPLC analysis (275 nm) of CreD catalyzed reactions. s1 and s2, authentic standards; i, 4-methylbenzyl phosphate as substrate; ii, benzylalcohol-4-phosphate as substrate; iii, benzylaldehyde-4-phosphate as substrate; iv, benzoate-4-phosphate as substrate. Traces in blue represent corresponding control reactions without addition of enzymes. Note that because of distinct extinction coefficients, the peak intensity of compounds do not necessarily reflect relative amounts.

FIGURE 6.

The catabolic pathway for 4-cresol from C. glutamicum. Bold arrows indicate the major phosphorylated pathway comprised of enzymatic steps with greater k_cat/K_m values. Each number in bracket denotes an individual enzymatic step.

FIGURE 7.

One-pot reaction analyses. A, HPLC analysis (275 nm) of the one-pot reaction containing all enzymes (CreHI, CreJEF, CreD, CreG, and CreC) under different molar ratios of 4-cresol:ATP. s, authentic standards; i–iv, ATP:4-cresol = 0, 1, 2, 3. B, HPLC analysis (275 nm) of the one-pot reaction with different enzyme combinations. s, authentic standards; i, all enzymes (CreHI, CreJEF, CreD, CreG, and CreC) in one pot; ii, all enzymes except CreC and CreG; iii, all enzymes except CreC; iv, all enzymes except CreG. All involved enzymes were normalized to the same concentration of 12.0 μm. All assays used 200 μm 4-cresol as substrate and were carried out in 100 mm Tris-HCl buffer (pH 8.0) at 30 °C for 120 min. In assays of B, the initial concentration of ATP was 600 μm, and all conversions of 4-cresol were complete. Note that each percentage indicates the conversion ratio from 4-cresol to the specific product. Because of distinct extinction coefficients, the peak intensity of compounds do not necessarily reflect relative amounts shown in percentage.