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Discovery, characterization and engineering of ligases for amide synthesis

Nature volume 593, pages 391–398 (2021)Cite this article

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Abstract

Coronatine and related bacterial phytotoxins are mimics of the hormone jasmonyl-l-isoleucine (JA-Ile), which mediates physiologically important plant signalling pathways1,2,3,4. Coronatine-like phytotoxins disrupt these essential pathways and have potential in the development of safer, more selective herbicides. Although the biosynthesis of coronatine has been investigated previously, the nature of the enzyme that catalyses the crucial coupling of coronafacic acid to amino acids remains unknown1,2. Here we characterize a family of enzymes, coronafacic acid ligases (CfaLs), and resolve their structures. We found that CfaL can also produce JA-Ile, despite low similarity with the Jar1 enzyme that is responsible for ligation of JA and l-Ile in plants5. This suggests that Jar1 and CfaL evolved independently to catalyse similar reactions—Jar1 producing a compound essential for plant development4,5, and the bacterial ligases producing analogues toxic to plants. We further demonstrate how CfaL enzymes can be used to synthesize a diverse array of amides, obviating the need for protecting groups. Highly selective kinetic resolutions of racemic donor or acceptor substrates were achieved, affording homochiral products. We also used structure-guided mutagenesis to engineer improved CfaL variants. Together, these results show that CfaLs can deliver a wide range of amides for agrochemical, pharmaceutical and other applications.

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Fig. 1: Biosynthesis of coronatine (COR) and jasmonyl-l-isoleucine (JA-Ile).
Fig. 2: Structure of PbCfaL.
Fig. 3: Carboxylic acid substrate scope.
Fig. 4: Amino acid substrate scope.
Fig. 5: Synthetic potential of CfaL enzymes.

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Data availability

Nucleotide sequences for the mutants generated as part of this study are available in Supplementary Information. Other nucleotide sequences for the enzymes used in this study were obtained from GenBank, and their accession numbers are provided within the paper or in Supplementary Information. The original materials and data that support the findings of this study are either available within the paper or are available from the corresponding author upon reasonable request. Crystallographic coordinates of wild type and mutant PbCfaL have been deposited in the Protein Data Bank as 7A9I (wild type) and 7A9J (R395G mutant).

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Acknowledgements

We thank the BBSRC (grants BB/K002341/1 and BB/N023536/1) and Syngenta for funding. F.W. was supported by the China Scholarship Council (grant no. 201806155100) and L.B. was funded by the Deutsche Forschungsgemeinschaft (DFG, grant BE 7054/1). The Michael Barber Centre for Collaborative Mass Spectrometry provided access to MS instrumentation. We also thank J. Vincent and N. Mulholland (Syngenta) for helpful discussions in the early stages of the project, and N. J. Turner (University of Manchester) for kindly providing the CHU plasmid. We also thank Diamond Light Source for beamtime access on i03 and i04-1 (proposal mx17773-56 and 76).

Author information

Author notes

  1. Fanghua Wang

    Present address: School of Food Science and Engineering, South China University of Technology, Guangzhou, China

Authors and Affiliations

  1. Department of Chemistry, Manchester Institute of Biotechnology, The University of Manchester, Manchester, UK

    Michael Winn, Michael Rowlinson, Fanghua Wang, Luis Bering, Daniel Francis, Colin Levy & Jason Micklefield

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Contributions

M.W. and J.M. designed experiments; M.W., M.R., F.W., L.B. and D.F. carried out the experiments and provided additional experiment design; C.L. performed crystallographic studies. M.W. and J.M. wrote the manuscript. J.M. led the study.

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Correspondence to Jason Micklefield.

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Extended data figures and tables

Extended Data Fig. 1 Melting point determination of CfaL enzymes.

Melting point temperatures (Tm) of the CfaLs in this study, obtained using a fluorescence-based assay conducted in a Bio-Rad CFX Connect qPCR machine. Higher Tm indicates improved thermal stability. The Tm is calculated as the lowest point when plotting the negative derivative of RFU (relative fluorescence units) as a function of temperature (dT), versus the temperature (degrees Celsius).

Extended Data Fig. 2 Rational mutagenesis of PbCfaL.

a, Structural comparison between PbCfaL (left) and the mutant PbCfaL(R395G) (right). R395 (circled) of PbCfaL (PDB ID 7A9I) is in the hinge region between the N-terminal domain (blue) and the flexible C-terminal domain (red). In PbCfaL(R395G) (PDB ID 7A9J) this large arginine residue is replaced by a much smaller glycine (circled) that is found in the other members of the CfaL family and many other similar ANL ligases. The overall structure of this mutant exhibits no other substantial structural difference from that of the wild type. b, Overlay of PbCfaL with three published ATP-dependent ligase structures (in ellipse) showing the conserved ATP binding location. When superimposed, PbCfaL (PDB ID 7A9I, blue), McbA (AMP bound, PDB ID 6SQ8, red), GrsA (ATP bound, PDB ID 1AMU, green) and AuaEII (anthranoyl-AMP bound, PDB ID 4WV3, light brown) show the conserved location of ATP binding. The corresponding loop in PbCfaL (inset, arrowed) is larger than in the other structures which may affect ATP binding. The location of this region within the structure of PbCfaL (grey) is also shown for reference. Structural alignment was performed using Chimera (version 1.14) MatchMaker.

Extended Data Fig. 3 Synthesis and NMR spectra of crude product 64.

a, Preparative-scale synthesis of 64 from 10 and l-Ile catalysed by PbCfaL(R395G/A294P) (lysate), with either the addition of ATP (87% isolated yield) or recycling the endogenous ATP present in the lysate using the kinase (CHU) and polyphosphate (polyP) (52% isolated yield). b, 1H-NMR spectrum of crude product 64. c, 13C-NMR spectrum of crude product 64.

Extended Data Fig. 4 Catalysed conversion of 10 and l-Ile to give 64.

a, Catalysed by PbCfaL (R395G/A294P) CLEAs. Reactions (100 mM Tris-HCl, 10 mM MgCl2, 10 mM ATP, 1 mM 10, 3 mM l-isoleucine, 50 ml total volume) were run for 24 h; the CLEA (cross-linked enzyme aggregate) was then removed, washed, and reintroduced to an identical reaction. While activity was seen to reduce over the 5 days, the CLEA still retained high levels of productivity even after 5 recycles over 5 days, whereas cell lysates generally precipitated and lost all activity within 12 h. Although CfaL undergoes extensive conformational changes during catalysis, encapsulating it within CLEAs shows the potential of immobilization to extend the functional lifespan of the CfaL. More sophisticated immobilization techniques may have the potential to further retain activity. Conversion values were calculated from HPLC peak area ratios of product and starting materials, and represent means where n = 5, error bars denote s.d. b, Catalysed by purified PbCfaL(R395G/A294P), showing percentage conversions of 10 and l-Ile in the presence of various solvents and at different concentrations. Conversion values were calculated from HPLC peak area ratios of product and starting materials and represent means where n = 3, error bars denote s.d.

Extended Data Fig. 5 LCMS analysis and extracted-ion chromatograms (EICs) of fragments of telaprevir and bortezomib synthesized by PbCfaL(R395G/A294P).

a, Proposed route towards the antiviral agent telaprevir by CfaL (see reaction at top). The expected product of the reaction, 110, was detected by LCMS (top trace, expected m/z 262.1197, observed 262.1193 [M-H]−). Additional peaks consistent with dipeptide 111, formed from condensation of two cyclohexylglycines, 58, were also detected (bottom trace, 111a and 111b, expected m/z 295.2027, observed 295.2036 [M-H]−). Although CfaL are highly selective for l-amino acid substrates, the appearance of two products of the same mass suggests formation of diastereomers, which may be due to a lack of enantioselectivity in the adenylation step forming the acyl donor when racemic cyclohexylglycine (58) is used. This indicates that 58 can function as both a carboxylic acid and an amine donor. b, Proposed route towards anti-cancer agent bortezomib via the synthesis of 112 by CfaL (see reaction at top). The expected product of the reaction was detected by LCMS (top trace, expected m/z 270.0884, observed 270.0878 [M-H]−). An additional peak consistent with an l-Phe dipeptide (113) was also detected (bottom trace, expected m/z 311.1401). This indicates that l-Phe can function as both acyl donor and amine acceptor. c, The reaction between carboxylic acid substrate (9), which is a good substrate for the enzyme, and cyclohexylglycine (58) gives only the desired product (114, top trace, expected m/z 274.1449, observed 274.1460 [M-H]−). No cyclohexylglycine homodimer (dipeptide 111) was evident in this case, indicating that homocoupling of 58 only takes place when carboxylic acid (acyl donor) substrates that are not well accepted by CfaL are used.

Extended Data Table 1 Table of kinetic parameters for adenylation reactions
Full size table
Extended Data Table 2 Percentage yield of ligation reactions of carboxylic acids
Full size table
Extended Data Table 3 Ligation of 9 to a range of proteinogenic amino acids
Full size table
Extended Data Table 4 Ligation of 9 to a range of non-proteinogenic amino acids
Full size table

Supplementary information

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Winn, M., Rowlinson, M., Wang, F. et al. Discovery, characterization and engineering of ligases for amide synthesis. Nature 593, 391–398 (2021). https://doi.org/10.1038/s41586-021-03447-w

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