Stereochemical Insights into the Anaerobic Degradation of 4‐Isopropylbenzoyl‐CoA in the Denitrifying Bacterium Strain pCyN1

Abstract

The constitutions and absolute configurations of two previously unknown intermediates, (1S,2S,4S)‐2‐hydroxy‐4‐isopropylcyclohexane‐1‐carboxylate and (S)‐3‐isopropylpimelate, of anaerobic degradation of p‐cymene in the bacterium Aromatoleum aromaticum pCyN1 are reported. These intermediates (as CoA esters) are involved in the further degradation of 4‐isopropylbenzoyl‐CoA formed by methyl group hydroxylation and subsequent oxidation of p‐cymene. Proteogenomics indicated 4‐isopropylbenzoyl‐CoA degradation involves (i) a novel member of class I benzoyl‐CoA reductase (BCR) as known from Thauera aromatica K172 and (ii) a modified β‐oxidation pathway yielding 3‐isopropylpimeloyl‐CoA analogously to benzoyl‐CoA degradation in Rhodopseudomonas palustris. Reference standards of all four diastereoisomers of 2‐hydroxy‐4‐isopropylcyclohexane‐1‐carboxylate as well as both enantiomers of 3‐isopropylpimelate were obtained by stereoselective syntheses via methyl 4‐isopropyl‐2‐oxocyclohexane‐1‐carboxylate. The stereogenic center carrying the isopropyl group was established using a rhodium‐catalyzed asymmetric conjugate addition. X‐ray crystallography revealed that the thermodynamically most stable stereoisomer of 2‐hydroxy‐4‐isopropylcyclohexane‐1‐carboxylate is formed during p‐cymene degradation. Our findings imply that the reductive dearomatization of 4‐isopropylbenzoyl‐CoA by the BCR of A. aromaticum pCyN1 stereospecifically forms (S)‐4‐isopropyl‐1,5‐cyclohexadiene‐1‐carbonyl‐CoA.

Complete Genomes of the Anaerobic Degradation Specialists <b><i>Aromatoleum petrolei</i></b> ToN1^T and <b><i>Aromatoleum bremense</i></b> PbN1^T

“…Notably, the genome of Ar. petrolei ToN1 T does not contain genes for a 4-isopropylbenzoyl-CoA-specific reductase [Küppers et al, 2019], agreeing with its incapacity to anaerobically degrade the aromatic monoterpene p-cymene [Rabus et al, 2019]. These differences in the genetic equipment and thereof resulting range of utilizable monoterpenes between Ar.…”

“…(iii) Modified β-oxidation yielding the respective pimeloyl-CoA derivate is then presumably conducted by the BadH-K enzymes as recently proposed for Ar. aromaticum pCyN1 [Küppers et al, 2019]. In case of limonene the exocyclic double bond is probably retained along the hypothesized pathway to the level of the pimeloyl-CoA derivate (Fig.…”

Complex and flexible catabolism in Aromatoleum aromaticum pCyN1

“…3). In addition to this peripheral module, protein components of the downstream conversion, viz., Thauera-type class I benzoyl-CoA reductase followed by Rhodopseudomonas-type β-oxidation (Küppers et al, 2019), also revealed a rather high specificity for these monoterpenes. This, in turn, indicates a concerted transcriptional regulation, even though the respective genes are scattered across the 'p-cymene' gene cluster (Fig.…”

A Straightforward Synthesis of Trideuterated α‐Terpinene for Mechanistic Studies

“…The presence of these compounds suggests a degradation pathway of α-terpinene (6a) that is similar to the known degradation pathway of p-cymene (1). [23,24] In addition, p-isopropylbenzoyl-CoA (4b) was abundant in the culture extracts (unpublished results; Rabus, Wilkes, Christoffers, and co-workers), which is not necessarily expected to occur as an intermediate during anaerobic degradation of α-terpinene (6a). Based on these findings and taking into account the possibility of a disproportionation reaction, a pathway for the degradation of α-terpinene (6a) is proposed (Scheme 1, steps e-p), including a disproportionation of 4-isopropyl-1,3-cyclohexadiene-1-carbonyl-CoA (9b) to p-isopropylbenzoyl-CoA (4b) and 4isopropyl-1-cyclohexene-1-carbonyl-CoA (10a) (Scheme 2, Equation 2).…”

“…After further functional group interconversions (steps f, m, n) the ring is cleaved to furnish 3-isopropylpimeloyl-CoA (11, step o). [23,24] Other investigations showed that the disproportionation of dienoyl-CoA 14 to benzoyl-CoA (16) and enoyl-CoA 15 is catalyzed by a tungsten-containing class II BCR under artificial conditions (Scheme 2, Equation 1) as well as by Ferroglobus placidus cell extracts by a so far unknown enzyme. [25,26] Latest investigations in our laboratories on methylated extracts of bac-terial cultures of strain pCyN1 grown on α-terpinene (6a) led to the identification of four compounds as their corresponding methyl esters, namely 4-isopropyl-1,5-cyclohexadiene-1-carbonyl-CoA (9b), 4-isopropyl-1-cyclohexene-1-carbonyl-CoA (10a), 2-hydroxy-4-isopropylcyclohexane-1-carbonyl-CoA (13) and 3isopropylpimeloyl-CoA (11) (unpublished results; Rabus, Wilkes, Christoffers and co-workers).…”