Biocatalytic Carboxylate Reduction – Recent Advances and New Enzymes

Winkler, Margit; Ling, Jonathan Guyang

doi:10.1002/cctc.202200441

Cited by 13 publications

(22 citation statements)

References 93 publications

(167 reference statements)

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“…CARs can be divided into five clans: The group of bacterial CARs belongs to type I, while type III consists of CARs from Ascomycetes, type IV of CARs from Basidiomycetes and type II represents a mixed group of fungal origin . The fifth type of enzymes of similar domain architecture is specialized in reducing aromatic amino acids . Sequence similarity between different CAR types is low, hence the structural insight from one clan is of limited value for the others.…”

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Structure of the Reductase Domain of a Fungal Carboxylic Acid Reductase and Its Substrate Scope in Thioester and Aldehyde Reduction

et al. 2022

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The synthesis of aldehydes from carboxylic acids has long been a challenge in chemistry. In contrast to the harsh chemically driven reduction, enzymes such as carboxylic acid reductases (CARs) are considered appealing biocatalysts for aldehyde production. Although structures of single- and didomains of microbial CARs have been reported, to date no full-length protein structure has been elucidated. In this study, we aimed to obtain structural and functional information regarding the reductase (R) domain of a CAR from the fungus Neurospora crassa (Nc). The NcCAR R-domain revealed activity for N-acetylcysteamine thioester (S-(2-acetamidoethyl) benzothioate), which mimics the phosphopantetheinylacyl-intermediate and can be anticipated as the minimal substrate for thioester reduction by CARs. The determined crystal structure of the NcCAR R-domain reveals a tunnel that putatively harbors the phosphopantetheinylacyl-intermediate, which is in good agreement with docking experiments performed with the minimal substrate. In vitro studies were performed with this highly purified R-domain and NADPH, demonstrating carbonyl reduction activity. The R-domain was able to accept not only a simple aromatic ketone but also benzaldehyde and octanal, which are typically considered to be the final product of carboxylic acid reduction by CAR. Also, the full-length NcCAR reduced aldehydes to primary alcohols. In conclusion, aldehyde overreduction can no longer be attributed exclusively to the host background.

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Structure of the Reductase Domain of a Fungal Carboxylic Acid Reductase and Its Substrate Scope in Thioester and Aldehyde Reduction

et al. 2022

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“…A total of 84 proteins from 28 fungal species were revealed as CARs with the typical A-T-R domain architecture ( Figure 2 ). 29 of them have been previously reported for carboxylic acid reduction by genome mining and in vitro enzyme assays [ 4 , 8 , 32 ]. Ten CARs containing a well-known AtCAR and nine other CARs were mined in A. terreus , which is consistent with a previous study [ 4 ].…”

Section: Resultsmentioning

confidence: 99%

“…Represented by Nps3 and Lys2, CARs in clade II are L-aminoadipate reductases [ 35 ]. In clade III, only NcCAR and TtCAR were reported with relaxed substrate tolerances for reducing aryl-aliphatic carboxylic acids, aliphatic carboxylic acids and a few heterocyclic aromatic carboxylic acids [ 8 ]. Fub8 from Fusarium fujikuroi catalyzes the reduction of trans -2-hexenoic acid in the biosynthesis of fusaric acid [ 36 ].…”

Section: Resultsmentioning

confidence: 99%

“…Enzymatic reduction of carboxylic acids via CARs was reported as earlier as 1959 for NcCAR characterization [ 9 ]. Genome mining and the techniques of genetic manipulation have led to approximately 50 identified CARs with a broad substrate scope for accepting aromatic, polycyclic and heteroaromatic carboxylic acids; aliphatic carboxylic acids; aryl-aliphatic carboxylic acids; and amino acids [ 8 ]. Inspired by the catalytic potential of CARs, we constructed a fungal virtual CAR library containing 84 enzymes with 55 new CARs from 28 species for the first time in this study.…”

Section: Discussionmentioning

confidence: 99%

“…Represented by ApvA, BtyA and AtqA identified from Aspergillus terreus , NRPS-like enzymes with an A-T-TE domain architecture catalyze the dimerization of α-keto acids, leading to the formation of lactones, ketals or benzoquinones [ 6 , 7 ]. Differing from the TE domain, the R domain in NRPS-like enzymes functions for carboxylic acid reduction to afford aldehyde with the help of upstream substrate adenylation by the A domain at the expense of ATP, thioester linkage formation by T domain and NADPH as a hydride donor ( Figure 1 A) [ 8 ]. Hence, NRPS-like enzymes containing A-T-R domain architecture are also known as carboxylate reductases (CARs).…”

Section: Introductionmentioning

confidence: 99%

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Characterization of a NRPS-like Protein from Pestalotiopsis fici for Aldehyde Generation

Wei

Ran

et al. 2022

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Nonribosomal peptide synthetase (NRPS)-like enzymes containing A-T-R domain architecture are also known as carboxylate reductases (CARs) for aldehyde generation. To identify new members of CARs, we established a virtual library containing 84 fungal CARs distributed in seven distinct clades by genome mining and phylogenetic analysis. Nine CARs, including PnlA from Pestalotiopsis fici and eight known CARs, were clustered in clade VI and proposed to catalyze the reduction of nonreducing polyketide synthase (NR-PKS)-derived aryl carboxylic acids. The recombinant protein PnlA was overproduced and purified to apparent homogeneity from Saccharomyces cerevisiae. In vitro enzyme assays of PnlA with 28 different benzoic acid derivatives (1–28) revealed the corresponding aldehyde formation in 14 cases (1–14). Comparison of conversion yields indicated the high preference of PnlA toward 3,5-dimethylorsellinic acid (DMOA, 4) and vanillic acid (10). A specificity-conferring code Q355 in PnlA was postulated by sequence alignment with the known CARs in clade VI. Our study provides an updated virtual library of fungal CAR enzymes and expands the biocatalytic selectivity of CARs.

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Organic Acid to Nitrile: A Chemoenzymatic Three‐Step Route

et al. 2022

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Various widely applied compounds contain cyano-groups, and this functional group serves as a chemical handle for a whole range of different reactions. We report a cyanide free chemoenzymatic cascade for nitrile synthesis. The reaction pathway starts with a reduction of carboxylic acid to aldehyde by carboxylate reductase enzymes (CARs) applied as living cell biocatalysts. The second -chemical -step includes in situ oxime formation with hydroxylamine. The final direct step from oxime to nitrile is catalyzed by aldoxime dehydratases (Oxds). With compatible combinations of a CAR and an Oxd, applied in one-pot two-step reactions, several aliphatic and aryl-aliphatic target nitriles were obtained in more than 80% conversion. Phenylacetonitrile, for example, was prepared in 78% isolated yield. This chemoenzymatic route does not require cyanide salts, toxic metals, or undesired oxidants in contrast to entirely chemical procedures.

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Biocatalytic Carboxylate Reduction – Recent Advances and New Enzymes

Cited by 13 publications

References 93 publications

Structure of the Reductase Domain of a Fungal Carboxylic Acid Reductase and Its Substrate Scope in Thioester and Aldehyde Reduction

Structure of the Reductase Domain of a Fungal Carboxylic Acid Reductase and Its Substrate Scope in Thioester and Aldehyde Reduction

Characterization of a NRPS-like Protein from Pestalotiopsis fici for Aldehyde Generation

Organic Acid to Nitrile: A Chemoenzymatic Three‐Step Route

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