A promiscuous coenzyme A ligase provides benzoyl-coenzyme A for xanthone biosynthesis in Hypericum.

Benzoic acid-derived compounds, such as polyprenylated benzophenones and xanthones, attract the interest of scientists due to challenging chemical structures and diverse biological activities. The genus Hypericum is of high medicinal value, as exemplified by H. perforatum. It is rich in benzophenone and xanthone derivatives, the biosynthesis of which requires the catalytic activity of benzoate-coenzyme A (benzoate-CoA) ligase (BZL), which activates benzoic acid to benzoyl-CoA. Despite remarkable research so far done on benzoic acid biosynthesis in planta, all previous structural studies of BZL genes and proteins are exclusively related to benzoate-degrading microorganisms. Here, a transcript for a plant acyl-activating enzyme (AAE) was cloned from xanthone-producing Hypericum calycinum cell cultures using transcriptomic resources. An increase in the HcAAE1 transcript level preceded xanthone accumulation after elicitor treatment, as previously observed with other pathway-related genes. Subcellular localization of reporter fusions revealed the dual localization of HcAAE1 to cytosol and peroxisomes owing to a type 2 peroxisomal targeting signal. This result suggests the generation of benzoyl-CoA in Hypericum by the CoA-dependent non-ß-oxidative route. A luciferase-based substrate specificity assay and the kinetic characterization indicated that HcAAE1 exhibits promiscuous substrate preference, with benzoic acid being the sole aromatic substrate accepted. Unlike 4-coumarate-CoA ligase and cinnamate-CoA ligase enzymes, HcAAE1 did not accept 4-coumaric and cinnamic acids, respectively. The substrate preference was corroborated by in silico modeling, which indicated valid docking of both benzoic acid and its adenosine monophosphate intermediate in the HcAAE1/BZL active site cavity.

Aryl Coenzyme A Ligases, a Subfamily of the Adenylate-Forming Enzyme Superfamily.

Aryl coenzyme A (CoA) ligases belong to class I of the adenylate-forming enzyme superfamily (ANL superfamily). They catalyze the formation of thioester bonds between aromatic compounds and CoA and occur in nearly all forms of life. These ligases are involved in various metabolic pathways degrading benzene, toluene, ethylbenzene, and xylene (BTEX) or polycyclic aromatic hydrocarbons (PAHs). They are often necessary to produce the central intermediate benzoyl-CoA that occurs in various anaerobic pathways. The substrate specificity is very diverse between enzymes within the same class, while the dependency on Mg2+, ATP, and CoA as well as oxygen insensitivity are characteristics shared by the whole enzyme class. Some organisms employ the same aryl-CoA ligase when growing aerobically and anaerobically, while others induce different enzymes depending on the environmental conditions. Aryl-CoA ligases can be divided into two major groups, benzoate:CoA ligase-like enzymes and phenylacetate:CoA ligase-like enzymes. They are widely distributed between the phylogenetic clades of the ANL superfamily and show closer relationships within the subfamilies than to other aryl-CoA ligases. This, together with residual CoA ligase activity in various other enzymes of the ANL superfamily, leads to the conclusion that CoA ligases might be the ancestral proteins from which all other ANL superfamily enzymes developed.

Biotransformation of Benzoate to 2,4,6-Trihydroxybenzophenone by Engineered Escherichia coli.

The synthesis of natural products by E. coli is a challenging alternative method of environmentally friendly minimization of hazardous waste. Here, we establish a recombinant E. coli capable of transforming sodium benzoate into 2,4,6-trihydroxybenzophenone (2,4,6-TriHB), the intermediate of benzophenones and xanthones derivatives, based on the coexpression of benzoate-CoA ligase from Rhodopseudomonas palustris (BadA) and benzophenone synthase from Garcinia mangostana (GmBPS). It was found that the engineered E. coli accepted benzoate as the leading substrate for the formation of benzoyl CoA by the function of BadA and subsequently condensed, with the endogenous malonyl CoA by the catalytic function of BPS, into 2,4,6-TriHB. This metabolite was excreted into the culture medium and was detected by the high-resolution LC-ESI-QTOF-MS/MS. The structure was elucidated by in silico tools: Sirius 4.5 combined with CSI FingerID web service. The results suggested the potential of the new artificial pathway in E. coli to successfully catalyze the transformation of sodium benzoate into 2,4,6-TriHB. This system will lead to further syntheses of other benzophenone derivatives via the addition of various genes to catalyze for functional groups.

The benzoyl-CoA pathway serves as a genomic marker to identify the oxygen requirements in the degradation of aromatic hydrocarbons.

The first step of anaerobic benzoate degradation is the formation of benzoyl-coenzyme A by benzoate-coenzyme A ligase (BCL). The anaerobic route is steered by benzoyl-CoA reductase, which promotes benzoyl-CoA breakdown, which is subsequently oxidized. In certain bacteria at low oxygen conditions, the aerobic metabolism of monoaromatic hydrocarbons occurs through the degradation Box pathway. These pathways have undergone experimental scrutiny in Alphaproteobacteria and Betaproteobacteria and have also been explored bioinformatically in representative Betaproteobacteria. However, there is a gap in our knowledge regarding the distribution of the benzoyl-CoA pathway and the evolutionary forces propelling its adaptation beyond that of representative bacteria. To address these questions, we used bioinformatic procedures to identify the BCLs and the lower pathways that transform benzoyl-CoA. These procedures included the identification of conserved motifs. As a result, we identified two motifs exclusive to BCLs, describing some of the catalytic properties of this enzyme. These motifs helped to discern BCLs from other aryl-CoA ligases effectively. The predicted BCLs and the enzymes of lower pathways were used as genomic markers for identifying aerobic, anaerobic, or hybrid catabolism, which we found widely distributed in Betaproteobacteria. Despite these enhancements, our approach failed to distinguish orthologs from a small cluster of paralogs exhibiting all the specified features to predict an ortholog. Nonetheless, the conducted phylogenetic analysis and the properties identified in the genomic context aided in formulating hypotheses about how this redundancy contributes to refining the catabolic strategy employed by these bacteria to degrade the substrates.