Structural studies reveal flexible roof of active site responsible for ω-transaminase CrmG overcoming by-product inhibition

Communications Biology - Tập 3 Số 1
Jinxin Xu1, Xiaowen Tang2, Yiguang Zhu3, Zhijun Yu1, Kai Su1, Yulong Zhang1, Yan Dong1, Weiming Zhu4, Changsheng Zhang3, Ruibo Wu2, Jinsong Liu5
1State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 510530, Guangzhou, China
2School of Pharmaceutical Sciences, Sun Yat-sen University, 510006 Guangzhou, China
3Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, 510301, Guangzhou, China
4Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, 266003, Qingdao, China
5Graduate University of Chinese Academy of Sciences, 100049 Beijing, China

Tóm tắt

AbstractAmine compounds biosynthesis using ω-transaminases has received considerable attention in the pharmaceutical industry. However, the application of ω-transaminases was hampered by the fundamental challenge of severe by-product inhibition. Here, we report that ω-transaminase CrmG from Actinoalloteichus cyanogriseus WH1-2216-6 is insensitive to inhibition from by-product α-ketoglutarate or pyruvate. Combined with structural and QM/MM studies, we establish the detailed catalytic mechanism for CrmG. Our structural and biochemical studies reveal that the roof of the active site in PMP-bound CrmG is flexible, which will facilitate the PMP or by-product to dissociate from PMP-bound CrmG. Our results also show that amino acceptor caerulomycin M (CRM M), but not α-ketoglutarate or pyruvate, can form strong interactions with the roof of the active site in PMP-bound CrmG. Based on our results, we propose that the flexible roof of the active site in PMP-bound CrmG may facilitate CrmG to overcome inhibition from the by-product.

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Tài liệu tham khảo

Gomm, A. & O’Reilly, E. Transaminases for chiral amine synthesis. Curr. Opin. Chem. Biol. 43, 106–112 (2018).

Malik, M. S., Park, E. S. & Shin, J. S. Features and technical applications of omega-transaminases. Appl. Microbiol. Biotechnol. 94, 1163–1171 (2012).

Hwang, B.-Y., Cho, B.-K., Yun, H., Koteshwar, K. & Kim, B.-G. Revisit of aminotransferase in the genomic era and its application to biocatalysis. J. Mol. Catal. B 37, 47–55 (2005).

Guo, F. & Berglund, P. Transaminase biocatalysis: optimization and application. Green Chem. 19, 333–360 (2017).

Kelly, S. A. et al. Application of omega-transaminases in the pharmaceutical industry. Chem. Rev. 118, 349–367 (2018).

Rocha, J. F., Pina, A. F., Sousa, S. F. & Cerqueira, N. M. F. S. A. PLP-dependent enzymes as important biocatalysts for the pharmaceutical, chemical and food industries: a structural and mechanistic perspective. Catal. Sci. Technol. 9, 4864–4876 (2019).

Mahesh, D. P., Grogan, G., Bommarius, A. & Yun, H. Recent advances in ω-transaminase-mediated biocatalysis for the enantioselective synthesis of chiral amines. Catalysts 8, 254 (2018).

Ferrandi, E. E. & Monti, D. Amine transaminases in chiral amines synthesis: recent advances and challenges. World J. Microbiol. Biotechnol. 34, 13 (2017).

Kelefiotis-Stratidakis, P., Tyrikos-Ergas, T. & Pavlidis, I. V. The challenge of using isopropylamine as an amine donor in transaminase catalysed reactions. Org. Biomol. Chem. 17, 1634–1642 (2019).

Koszelewski, D. et al. Formal asymmetric biocatalytic reductive amination. Angew. Chem. Int. Ed. Engl. 47, 9337–9340 (2008).

Shin, J. S. & Kim, B. G. Asymmetric synthesis of chiral amines with omega-transaminase. Biotechnol. Bioeng. 65, 206–211 (1999).

Koszelewski, D., Lavandera, I., Clay, D., Rozzell, D. & Kroutil, W. Asymmetric synthesis of optically pure pharmacologically relevant amines employing ω‐transaminases. Adv. Synth. Catal. 350, 2761–2766 (2008).

Savile, C. K. et al. Biocatalytic asymmetric synthesis of chiral amines from ketones applied to sitagliptin manufacture. Science 329, 305–309 (2010).

Han, S.-W., Park, E.-S., Dong, J.-Y. & Shin, J.-S. Mechanism-guided engineering of ω-transaminase to accelerate reductive amination of ketones. Adv. Synth. Catal. 357, 1732–1740 (2015).

Yun, H., Hwang, B. Y., Lee, J. H. & Kim, B. G. Use of enrichment culture for directed evolution of the Vibrio fluvialis JS17 omega-transaminase, which is resistant to product inhibition by aliphatic ketones. Appl. Environ. Microbiol 71, 4220–4224 (2005).

Park, E. S. & Shin, J. S. omega-Transaminase from Ochrobactrum anthropi is devoid of substrate and product inhibitions. Appl. Environ. Microbiol 79, 4141–4144 (2013).

Zhu, Y. et al. Identification of caerulomycin A gene cluster implicates a tailoring amidohydrolase. Org. Lett. 14, 2666–2669 (2012).

Zhu, Y. G. et al. Insights into caerulomycin a biosynthesis: a two-component monooxygenase CrmH-catalyzed oxime formation. J. Am. Chem. Soc. 135, 18750–18753 (2013).

Fu, P. et al. Acyclic congeners from actinoalloteichus cyanogriseus provide insights into cyclic bipyridine glycoside formation. Org. Lett. 16, 4264–4267 (2014).

Zhu, Y. G. et al. Flavoenzyme CrmK-mediated substrate recycling in caerulomycin biosynthesis. Chem. Sci. 7, 4867–4874 (2016).

Zhu, Y. et al. Biochemical and Structural Insights into the aminotransferase CrmG in Caerulomycin biosynthesis. ACS Chem. Biol. 11, 943–952 (2016).

Humble, M. S. et al. Crystal structures of the Chromobacterium violaceum ω-transaminase reveal major structural rearrangements upon binding of coenzyme PLP. FEBS J. 279, 779–792 (2012).

Newman, J. et al. Determination of the structure of the catabolic N-succinylornithine transaminase (AstC) from Escherichia coli. PLoS ONE 8, e58298 (2013).

Ruggieri, F. et al. Insight into the dimer dissociation process of the Chromobacterium violaceum (S)-selective amine transaminase. Sci. Rep. 9, 16946 (2019).

Shin, J. S. & Kim, B. G. Exploring the active site of amine:pyruvate aminotransferase on the basis of the substrate structure-reactivity relationship: how the enzyme controls substrate specificity and stereoselectivity. J. Org. Chem. 67, 2848–2853 (2002).

Schell, U., Wohlgemuth, R. & Ward, J. M. Synthesis of pyridoxamine 5’-phosphate using an MBA:pyruvate transaminase as biocatalyst. J. Mol. Catal. B 59, 279–285 (2009).

Manta, B., Cassimjee, K. E. & Himo, F. Quantum chemical study of dual-substrate recognition in omega-transaminase. ACS Omega 2, 890–898 (2017).

Cassimjee, K. E., Manta, B. & Himo, F. A quantum chemical study of the omega-transaminase reaction mechanism. Org. Biomol. Chem. 13, 8453–8464 (2015).

Han, S. W., Kim, J., Cho, H. S. & Shin, J. S. Active site engineering of co-transaminase guided by docking orientation analysis and virtual activity screening. ACS Catal. 7, 3752–3762 (2017).

Zhang, W.-Z. et al. The protein complex crystallography beamline (BL19U1) at the Shanghai Synchrotron Radiation Facility. Nucl. Sci. Technol. 30, 170 (2019).

Wang, Q.-S. et al. Upgrade of macromolecular crystallography beamline BL17U1 at SSRF. Nucl. Sci. Technol. 29, 68 (2018).

Battye, T. G., Kontogiannis, L., Johnson, O., Powell, H. R. & Leslie, A. G. iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr. D Biol. Crystallogr. 67, 271–281 (2011).

Winter, G. et al. DIALS: implementation and evaluation of a new integration package. Acta Crystallogr. D Struct. Biol. 74, 85–97 (2018).

Evans, P. R. An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr. D Biol. Crystallogr. 67, 282–292 (2011).

Vagin, A. & Teplyakov, A. MOLREP: an automated program for molecular replacement. J. Appl. Crystallogr. 30, 1022–1025 (1997).

Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367 (2011).

Case D. A. et al. AMBER 12 (University of California, San Francisco, 2012).

Duan, Y. et al. A point-charge force field for molecular mechanics simulations of proteins based on condensed-phase quantum mechanical calculations. J. Comput Chem. 24, 1999–2012 (2003).

Zhang, Y. K., Liu, H. Y. & Yang, W. T. Free energy calculation on enzyme reactions with an efficient iterative procedure to determine minimum energy paths on a combined ab initio QM/MM potential energy surface. J. Chem. Phys. 112, 3483–3492 (2000).

Shao, Y. et al. Advances in methods and algorithms in a modern quantum chemistry program package. Phys. Chem. Chem. Phys. 8, 3172–3191 (2006).

Rackers, J. A. et al. Tinker 8: software tools for molecular design. J. Chem. Theory Comput. 14, 5273–5289 (2018).

Zhang, F., Chen, N., Zhou, J. & Wu, R. Protonation-dependent diphosphate cleavage in FPP cyclases and synthases. ACS Catal. 6, 6918–6929 (2016).

Chen, N., Wang, S., Smentek, L., Hess, B. A. Jr. & Wu, R. Biosynthetic mechanism of lanosterol: cyclization. Angew. Chem. Int. Ed. Engl. 54, 8693–8696 (2015).

Zhang, F. et al. Enzyme promiscuity versus fidelity in two sesquiterpene cyclases (TEAS versus ATAS). ACS Catal. 10, 1470–1484 (2020).

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Communications Biology

Tập 3 Số 1

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