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 do...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
BMC
2019-07-01
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| Series: | Journal of Biological Engineering |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13036-019-0195-y |
| _version_ | 1828824240931995648 |
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| author | Fuchao Xu Russell Butler Kyle May Megi Rexhepaj Dayu Yu Jiachen Zi Yi Chen Yonghong Liang Jia Zeng Joan Hevel Jixun Zhan |
| author_facet | Fuchao Xu Russell Butler Kyle May Megi Rexhepaj Dayu Yu Jiachen Zi Yi Chen Yonghong Liang Jia Zeng Joan Hevel Jixun Zhan |
| author_sort | Fuchao Xu |
| collection | DOAJ |
| description | 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 (MTBSLS) and as part of the full NRPS megaenzyme. Surprisingly, the MTBSLS 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, MTBSLS, cMT, XLcMT, and aMT) to L-Phe-SNAC and L-Leu-SNAC, we further showed that restricting and/or fixing the termini of the MTBSLS 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. |
| first_indexed | 2024-12-12T13:52:43Z |
| format | Article |
| id | doaj.art-cf16e281bc28487cb84afd137f200ae6 |
| institution | Directory Open Access Journal |
| issn | 1754-1611 |
| language | English |
| last_indexed | 2024-12-12T13:52:43Z |
| publishDate | 2019-07-01 |
| publisher | BMC |
| record_format | Article |
| series | Journal of Biological Engineering |
| spelling | doaj.art-cf16e281bc28487cb84afd137f200ae62022-12-22T00:22:32ZengBMCJournal of Biological Engineering1754-16112019-07-0113111410.1186/s13036-019-0195-yModified substrate specificity of a methyltransferase domain by protein insertion into an adenylation domain of the bassianolide synthetaseFuchao Xu0Russell Butler1Kyle May2Megi Rexhepaj3Dayu Yu4Jiachen Zi5Yi Chen6Yonghong Liang7Jia Zeng8Joan Hevel9Jixun Zhan10Department of Biological Engineering, Utah State UniversityDepartment of Chemistry and Biochemistry, Utah State UniversityDepartment of Chemistry and Biochemistry, Utah State UniversityDepartment of Chemistry and Biochemistry, Utah State UniversityDepartment of Biological Engineering, Utah State UniversityDepartment of Biological Engineering, Utah State UniversityDepartment of Biological Engineering, Utah State UniversityDepartment of Biological Engineering, Utah State UniversityDepartment of Biological Engineering, Utah State UniversityDepartment of Chemistry and Biochemistry, Utah State UniversityDepartment of Biological Engineering, Utah State UniversityAbstract 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 (MTBSLS) and as part of the full NRPS megaenzyme. Surprisingly, the MTBSLS 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, MTBSLS, cMT, XLcMT, and aMT) to L-Phe-SNAC and L-Leu-SNAC, we further showed that restricting and/or fixing the termini of the MTBSLS 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.http://link.springer.com/article/10.1186/s13036-019-0195-yNonribosomal peptide synthetaseMethyltransferase domainSubstrate specificityDomain removalHeterologous expressionIn vitro reaction |
| spellingShingle | Fuchao Xu Russell Butler Kyle May Megi Rexhepaj Dayu Yu Jiachen Zi Yi Chen Yonghong Liang Jia Zeng Joan Hevel Jixun Zhan Modified substrate specificity of a methyltransferase domain by protein insertion into an adenylation domain of the bassianolide synthetase Journal of Biological Engineering Nonribosomal peptide synthetase Methyltransferase domain Substrate specificity Domain removal Heterologous expression In vitro reaction |
| title | Modified substrate specificity of a methyltransferase domain by protein insertion into an adenylation domain of the bassianolide synthetase |
| title_full | Modified substrate specificity of a methyltransferase domain by protein insertion into an adenylation domain of the bassianolide synthetase |
| title_fullStr | Modified substrate specificity of a methyltransferase domain by protein insertion into an adenylation domain of the bassianolide synthetase |
| title_full_unstemmed | Modified substrate specificity of a methyltransferase domain by protein insertion into an adenylation domain of the bassianolide synthetase |
| title_short | Modified substrate specificity of a methyltransferase domain by protein insertion into an adenylation domain of the bassianolide synthetase |
| title_sort | modified substrate specificity of a methyltransferase domain by protein insertion into an adenylation domain of the bassianolide synthetase |
| topic | Nonribosomal peptide synthetase Methyltransferase domain Substrate specificity Domain removal Heterologous expression In vitro reaction |
| url | http://link.springer.com/article/10.1186/s13036-019-0195-y |
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