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

Abstract

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.

Keywords: NRPS-like enzyme; Pestalotiopsis fici; aldehyde generation; benzoic acid derivatives; fungal carboxylate reductase (CAR).

Figure 1

Carboxylic acid reduction by CARs. (A) CARs containing A-T-R domain architecture catalyze two-electron reduction of carboxylates to aldehydes. (B) Representative fungal NPs involving carboxylic acid reduction by CARs in their biosynthetic pathways. They include cichorine from Aspergillus nidulans [19], LL-Z1272b from Stachybotrys bisbyi [13], epicospirocin A from Epicoccum nigrum [14], melanin derivative from Ustilago maydis [20], ascofuranone from Acremonium egyptiacum [21], aspergillic acid from Aspergillus flavus [22], actinopolymorphol C from A. flavus [15] and brasilliamide A from Penicillium brasilianum [16].

Scheme 1

Mechanism of the PnlA-catalyzed reduction of aryl carboxylic acid derivatives. Fourteen aryl carboxylic acid derivatives (1–14) were converted to the corresponding aldehydes (1′–14′).

Figure 2

Phylogenetic analysis of 84 CAR protein sequences from fungi. Seven distinct clades are shown in different colors. CARs in clade I, II and VI are mainly responsible for the reduction of amino acids, L-aminoadipate and orsellinic acid derivatives, respectively. CmlP characterized in Streptomyces venezuelae was used as the outgroup to root the tree [38]. Multiple alignments were performed by ClustalW with MEGA 7.0. The phylogenetic tree was constructed based on maximum likelihood (ML) analysis using RAxML 8.2.10 and visualized in iTOL v6. Amino acid sequences were obtained from the GenBank database.

Figure 3

LC-MS analyses of the incubation mixtures of PnlA with different aryl carboxylic acid derivatives (1–14). 1′–14′ are the corresponding aldehyde products. UV absorptions at 280 nm are illustrated for the enzyme assays of PnlA with 1–13, and those at 340 nm are illustrated for the enzyme assay of PnlA with 14. CK is the reaction mixture with denatured PnlA. Additional data on the enzyme assays of PnlA with 15–28 are presented in Figure S2.

Figure 4

Alignment of fungal CARs located in clade VI, including PnlA, StbB [13], AscB [21], CicB [19], Esp4 [14], AtCAR [17], PMAA_062890, ATEG_07380 and Pks5 [20]. See alignment of the whole sequences in Figure S5.