Determinants for Substrate Recognition in the Glycyl Radical Enzyme Benzylsuccinate Synthase Revealed by Targeted Mutagenesis

Abstract

Anaerobic toluene degradation is initiated by adding toluene to fumarate via the glycyl radical enzyme benzylsuccinate synthase (BSS). We developed a recombinant production system producing active BSS and present data on the effects of some active-site mutants. A single substitution of Ile617 to Val inside a hydrophobic pocket of the active site leads to an extended substrate range of the variant, which includes conversion of m-xylene, but not of o- or p-xylene. Moreover, a mutation of the conserved Arg508, which participates in fumarate binding, to Lys or Gln decreases or eliminates BSS activity, proving its catalytic importance. Surprisingly, the Arg508Lys mutant gained the ability to synthesize a small amount of the product 3-benzyl-4-ketopentanoate from toluene and the fumarate analogue 3-acetyl acrylate. This study provides insight into substrate recognition by BSS and opens avenues for possible broader biotechnological applications in C–C bond formation.

A tungsten enzyme using hydrogen as an electron donor to reduce carboxylic acids and NAD+

“…We used two plasmids for recombinant AOR Aa production, containing either aorABC or aorABCDE (see SI). The genes encoding the AOR Aa protein from A. aromaticum (genes ebA5004-5010) were obtained from chromosomal DNA by PCR ampli cation (details in SI) and ligated into a mobilizable vector based on the broad range vector pBBR 38,39 . The obtained plasmids were transformed into a conjugation strain of Escherichia coli (e.g.…”

“…The nal plasmid used contained the genes for the three subunits of AOR Aa (AorABC: WP_011238651 -WP_011238653) and the putative maturation factors AorD and AorE (WP_041646405 and WP_041646407) behind an anhydrotetracycline (AHT)-inducible promotor with an N-terminal Twin-Strep-tag fused to AorA. The plasmid was then transferred from the donor E. coli strain to A. evansii by conjugation 39 . The recombinant A. evansii strain was grown anaerobically on a minimal medium (pH 7.8) with benzoate and nitrate as described in Salii et al 39 , with the addition of ampicillin and Na 2 WO 4 to nal concentrations of 100 µg/mL and 18 nM, respectively.…”

“…The plasmid was then transferred from the donor E. coli strain to A. evansii by conjugation 39 . The recombinant A. evansii strain was grown anaerobically on a minimal medium (pH 7.8) with benzoate and nitrate as described in Salii et al 39 , with the addition of ampicillin and Na 2 WO 4 to nal concentrations of 100 µg/mL and 18 nM, respectively.…”

Engineering Escherichia coli for anaerobic alkane activation: Biosynthesis of (1‐methylalkyl)succinates

“…Metabolite profiling from pure cultures and environmental samples shows that the alkylsuccinates are further degraded via carbon‐skeleton rearrangement followed by decarboxylation and β‐oxidation (Kuppers et al, 2020) and fumarate regeneration; a representative pathway is depicted in Figure 1. To the best of our knowledge, there is no report of functional, heterologous expression of an alkylsuccinate synthase or corresponding activase (attempts to functionally express BSS proteins in Escherichia coli have proven unsuccessful (Salii et al, 2021)).…”

Protein Dynamics and Substrate Protonation State Mediate the Catalytic Action of Trans-4-Hydroxy-L-Proline Dehydratase

“…to reveal the structural determinants of benzylsuccinate synthase and its mutants for substrate recognition. 30 Our previous studies combined large-scale quantum mechanical calculations with steered molecular dynamics 31 to investigate the catalytic mechanisms of CutC, revealing the spontaneous decomposition of choline to trimethylamine subsequent to substrate C-H activation. 32 In contrast to other GRE 1,2-eliminases that involve the cleavage of a C-O or C-S bond in conformationally flexible substrates, 16 HypD catalyzes the oxidation of a C-N bond in a conformationally constrained pyrrolidine ring.…”