The molecular basis for the intramolecular migration (NIH shift) of the carboxyl group during para‐hydroxybenzoate catabolism

Abstract

The NIH shift is a chemical rearrangement in which a substituent on an aromatic ring undergoes an intramolecular migration, primarily during an enzymatic hydroxylation reaction. The molecular mechanism for the NIH shift of a carboxyl group has remained a mystery for 40 years. Here, we elucidate the molecular mechanism of the reaction in the conversion of para-hydroxybenzoate (PHB) to gentisate (GA, 2, 5-dihydroxybenzoate). Three genes (phgABC) from the PHB utilizer Brevibacillus laterosporus PHB-7a encode enzymes (p-hydroxybenzoyl-CoA ligase, p-hydroxybenzoyl-CoA hydroxylase and gentisyl-CoA thioesterase, respectively) catalyzing the conversion of PHB to GA via a route involving CoA thioester formation, hydroxylation concomitant with a 1, 2-shift of the acetyl CoA moiety and thioester hydrolysis. The shift of the carboxyl group was established rigorously by stable isotopic experiments with heterologously expressed phgABC, converting 2, 3, 5, 6-tetradeutero-PHB and [carboxyl- C]-PHB to 3, 4, 6-trideutero-GA and [carboxyl- C]-GA respectively. This is distinct from the NIH shifts of hydrogen and aceto substituents, where a single oxygenase catalyzes the reaction without the involvement of a thioester. The discovery of this three-step strategy for carboxyl group migration reveals a novel role of the CoA thioester in biochemistry and also illustrates the diversity and complexity of microbial catabolism in the carbon cycle.

A comparison between the homocyclic aromatic metabolic pathways from plant-derived compounds by bacteria and fungi

“…In principle, this conversion can be catalyzed by one enzyme through an intramolecular migration (NIH shift) of the carboxyl group, however it was never demonstrated for p-hydroxybenzoic acid. Pathway 8a was recently demonstrated in the bacterium Brevibacillus laterosporus that phydroxybenzoic acid converted to gentisic acid involving the three enzymes, p-hydroxybenzoyl-CoA ligase, p-hydroxybenzoyl-CoA hydroxylase and gentisyl-CoA thioesterase (Zhao et al, 2018). Pathway 8b was suggested in Rhizobium sp.…”

“…and Stryptomyces spp., gentisic acid is converted through extradiol ring cleavage to maleylpyruvate catalyzed by the gentisate 1,2-dioxygenase (SdgD or BagI) (Fig. 11) (Clark and Buswell, 1979;Ishiyama et al, 2004;Liu and Zhou et al, 2012;Sutherland et al, 1981;Zhao et al, 2018). Maleylpyruvate is then further converted to fumarylpyruvate by L-cysteine-dependent maleylpyruvate isomerase (BagL) that is further hydrolyzed by fumarylpyruvate hydrolase (BagK) into fumarate and pyruvate that enter the TCA cycle (Evans, 1963;Liu and Zhou et al, 2012).…”

Unveiling the CoA mediated salicylate catabolic mechanism in Rhizobium sp. X9

“…The enzyme assays were designed based on the previously published method (Ishiyama et al., 2004; Zhao et al., 2018). The standard enzyme reactions for salicylyl‐CoA ligase CehG were performed at 30℃ for 1 min in 1 ml of 20 mM glycine‐NaOH buffer (pH 9.0) containing salicylate (150 μM), ATP (150 μM), CoASH (150 μM), purified CehG (5 μg), and MgCl 2 (5 mM).…”

A Nag‐like dioxygenase initiates 3,4‐dichloronitrobenzene degradation via 4,5‐dichlorocatechol in Diaphorobacter sp. strain JS3050

“…For further determination of kinetic parameters the DcnC expressed by E . coli BL21(λDE3) [pET28a‐His dcnC ] was purified as described previously (Zhao et al ., 2018). A single colony of E .…”