Characterization and Engineering of the Adenylation Domain of a NRPS-Like Protein: A Potential Biocatalyst for Aldehyde Generation

Abstract

The adenylation (A) domain acts as the first “gate-keeper” to ensure the activation and thioesterification of the correct monomer to nonribosomal peptide synthetases (NRPSs). Our understanding of the specificity-conferring code and our ability to engineer A domains are critical for increasing the chemical diversity of nonribosomal peptides (NRPs). We recently discovered a novel NRPS-like protein (ATEG_03630) that can activate 5-methyl orsellinic acid (5-MOA) and reduce it to 2,4-dihydroxy-5,6-dimethyl benzaldehyde. A NRPS-like protein is much smaller than multidomain NRPSs, but it still represents the thioesterification half-reaction, which is otherwise missed from a stand-alone A domain. Therefore, a NRPS-like protein may serve as a better model system for A domain engineering. Here, we characterize the substrate specificity of ATEG_03630 and conclude that the hydrogen-bond donor at the 4-position is crucial for substrate recognition. Next, we show that the substrate specificity of ATEG_03630 can be engineered toward our target substrate anthranilate via bioinformatics analysis and mutagenesis. The resultant mutant H358A increased its activity toward anthranilate by 10.9-fold, which led to a 26-fold improvement in specificity. Finally, we demonstrate one-pot chemoenzymatic synthesis of 4-hydroxybenzaldoxime from 4-hydroxybenzoic acid with high yield.

Selective Enzymatic Transformation to Aldehydes in vivo by Fungal Carboxylate Reductase from Neurospora crassa

“…With the aim to identify the primary sequence of Neurospora crassa CAR, four literature known CAR enzyme sequences ( Ni CAR:5 Q6RKB1.1, Mm CAR:6 B2HN69, Sr CAR:8 WP_013138593.1 and At CAR:7 XP_001212808.1) were used as templates for a blastp search against the Gen‐Bank non‐redundant protein sequences of Neurospora crassa (taxid:5241). The hit with highest scores was a hypothetical protein with NCBI accession code XP_955820.1.…”

“…Notably, Nc CAR was expressed very efficiently in E. coli as a soluble protein, in contrast to another fungal CAR from Trametes versicolor under identical conditions 10. Other published fungal CARs ATEG03630 ( An CAR) and StbB ( Sb CAR) have been heterologously expressed in S. cerevisiae 7 and Aspergillus oryzae ,12 respectively. …”

“…Carboxylate reductase (CAR) enzymes exhibit a broad substrate tolerance for the conversion of organic acids to the respective aldehydes 4. However, only few CAR enzyme sequences have been elucidated5, 6, 7, 8, 9, 10, 11, 12 and are available for biocatalysis to date, although Nature provides a great versatility of organisms with the capability to catalyze this reaction. [4,13–18] The reduction of salicylic acid,19 benzoic acid and derivatives20 as well as cinnamic acid and derivatives21 has been observed in the ascomycete Neurospora crassa ( Nc ).…”

Exploring the synthetic applicability of a new carboxylic acid reductase from Segniliparus rotundus DSM 44985

“…1), which may be the rate-limiting step in the whole reaction. The N-terminal domain of CAR is responsible for adenylation and thioesterification of carboxylic acid, and it may play a role in determining the substrate specificity, which is similar to the adenylation domain of NRPS [28]. No reaction was observed when apo-CAR was used in the reaction system (23a as substrate) or the reaction system did not contain Mg 2+ (Supplementary Table T1), indicating that post-translational phosphopantetheinylation and Mg 2+ , ATP, and NADPH as cofactors were necessary for this enzymatic reduction.…”

An Engineered Aryl Acid Adenylation Domain with an Enlarged Substrate Binding Pocket

“…Forexample,anthranilic acid (Ant), p-aminobenzoic acid, and 3-hydroxypicolic acid are observed in acetylaszonalenin, [11] albicidin, [12] and pyridomycin, [13] respectively.O ur knowledge about the non-ribosomal codes for aryl acid building blocks is much more limited than for amino acid building blocks.E ngineering efforts focusing on aryl acid Adomains are therefore limited so far.The recognition properties of DhbE, an aryl acid A-domain from the siderophore bacillibactin biosynthetic pathway,h as been manipulated from DHB toward Ant with a2 06-fold specificity switch through directed evolution based on yeast-display technology. [15] Aryl acid A-domains frequently display the ability to catalyze adenylation toward ar ange of structurally related aryl acids. [15] Aryl acid A-domains frequently display the ability to catalyze adenylation toward ar ange of structurally related aryl acids.…”