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

Fuchao Xu; Russell Butler; Kyle May; Megi Rexhepaj; Dayu Yu; Jiachen Zi; Yi Chen; Yonghong Liang; Jia Zeng; Joan Hevel; Jixun Zhan

doi:10.1186/s13036-019-0195-y

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

J Biol Eng. 2019 Jul 31:13:65. doi: 10.1186/s13036-019-0195-y. eCollection 2019.

Authors

Fuchao Xu¹, Russell Butler², Kyle May², Megi Rexhepaj², Dayu Yu^{1

3}, Jiachen Zi¹, Yi Chen¹, Yonghong Liang^{1

4}, Jia Zeng¹, Joan Hevel², Jixun Zhan¹

Affiliations

¹ 1Department of Biological Engineering, Utah State University, 4105 Old Main Hill, Logan, UT 84322-4105 USA.
² 2Department of Chemistry and Biochemistry, Utah State University, 0300 Old Main Hill, Logan, UT 84322-0300 USA.
³ 3Department of Applied Chemistry and Biological Engineering, College of Chemical Engineering, Northeast Electric Power University, Jilin, 132012 Jilin China.
⁴ 4Key Laboratory of Modern Preparation of TCM, Ministry of Education, Jiangxi University of Traditional Chinese Medicine, Nanchang, 330004 Jiangxi China.

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.

Keywords: Domain removal; Heterologous expression; In vitro reaction; Methyltransferase domain; Nonribosomal peptide synthetase; Substrate specificity.