Modified substrate specificity of a methyltransferase domain by protein insertion into an adenylation domain of the bassianolide synthetase

Abstract

Background Creating designer molecules using a combination of select domains from polyketide synthases and/or nonribosomal peptide synthetases (NRPS) continues to be a synthetic goal. However, an incomplete understanding of how protein-protein interactions and dynamics affect each of the domain functions stands as a major obstacle in the field. Of particular interest is understanding the basis for a class of methyltransferase domains (MT) that are found embedded within the adenylation domain (A) of fungal NRPS systems instead of in an end-to-end architecture. Results The MT domain from bassianolide synthetase (BSLS) was removed and the truncated enzyme BSLS-ΔMT was recombinantly expressed. The biosynthesis of bassianolide was abolished and N -desmethylbassianolide was produced in low yields. Co-expression of BSLS-ΔMT with standalone MT did not recover bassianolide biosynthesis. In order to address the functional implications of the protein insertion, we characterized the N -methyltransferase activity of the MT domain as both the isolated domain (MT BSLS ) and as part of the full NRPS megaenzyme. Surprisingly, the MT BSLS construct demonstrated a relaxed substrate specificity and preferentially methylated an amino acid (L-Phe-SNAC) that is rarely incorporated into the final product. By testing the preference of a series of MT constructs (BSLS, MT BSLS , cMT, XLcMT, and aMT) to L-Phe-SNAC and L-Leu-SNAC, we further showed that restricting and/or fixing the termini of the MT BSLS by crosslinking or embedding the MT within an A domain narrowed the substrate specificity of the methyltransferase toward L-Leu-SNAC, the preferred substrate for the BSLS megaenzyme. Conclusions The embedding of MT into the A2 domain of BSLS is not required for the product assembly, but is critical for the overall yields of the final products. The substrate specificity of MT is significantly affected by the protein context within which it is present. While A domains are known to be responsible for selecting and activating the biosynthetic precursors for NRPS systems, our results suggest that embedding the MT acts as a secondary gatekeeper for the assembly line. This work thus provides new insights into the embedded MT domain in NRPSs, which will facilitate further engineering of this type of biosynthetic machinery to create structural diversity in natural products. Electronic supplementary material The online version of this article (10.1186/s13036-019-0195-y) contains supplementary material, which is available to authorized users.

Characterization of a Unique Interrupted Adenylation Domain That Can Catalyze Three Reactions

“…A series of radiometric and MS/MS assays demonstrated that the A, M s , and M b domains of ColG­(AM s M b A) function as we had hypothesized. The A domain specifically adenylated l -Ser, the M s domain (closest to the N-terminus of the ColG­(AM s M b A) enzyme) methylated the oxygen on the side chain of l -Ser and not the sulfur of l -Cys, and the M b domain (closest to the C-terminus of the ColG­(AM s M b A) enzyme) mono/dimethylated the nitrogen on the backbone of l -Ser as well as l -Cys and its methylated derivatives, as consistent with the relaxed specificity observed in other backbone M domains. ,, …”

“…The A domain specifically adenylated L-Ser, the M s domain (closest to the N-terminus of the ColG(AM s M b A) enzyme) methylated the oxygen on the side chain of L-Ser and not the sulfur of L-Cys, and the M b domain (closest to the C-terminus of the ColG(AM s M b A) enzyme) mono/dimethylated the nitrogen on the backbone of L-Ser as well as L-Cys and its methylated derivatives, as consistent with the relaxed specificity observed in other backbone M domains. 14,33,34 Nature continuously surprises us with new ways to introduce diversity in NPs and inspires us to create new ways to hopefully, in the future, produce biologically active "unnatural" natural products. The insights gained in this study open a variety of avenues for enzyme engineering and combinatorial biosynthesis studies for eventual drug discovery.…”

Repurposing Modular Polyketide Synthases and Non-ribosomal Peptide Synthetases for Novel Chemical Biosynthesis

“…This domain is usually integrated inside of A domain such as MT domain of cyclosporine A NRPS (Velkov et al, 2011). A recent engineering example of the MT domain in module 2 of bassianolide synthetase showed that the deletion of this domain generated the N-desmethylbassianolide without affecting the enzyme assembly lines (Xu et al, 2019). In another study, O-methylating MT domain was inserted between the A subdomains, resulting in the incorporation of O or N-methylated serine (Lundy et al, 2018).…”

Structures and function of a tailoring oxidase in complex with a nonribosomal peptide synthetase module

“…However, from the sequence of a BGC it is difficult to predict whether a separately encoded enzyme will act on an acyl substrate before assembly-line synthesis, on a PCP-tethered intermediate of NRPS assembly-line synthesis, or on a small molecule after release from the NRPS. The natures of these possible substrates are quite different, and an enzyme will act on only one of these species 24 , 32 .…”