Active site remodelling of a cyclodipeptide synthase redefines substrate scope

Funding: Wellcome Trust (210486/Z/18/Z), Cunningham Trust (PhD-CT-18-41).Cyclodipeptide synthases (CDPSs) generate a wide range of cyclic dipeptides using aminoacylated tRNAs as substrates. Histidine-containing cyclic dipeptides have important biological activities as anticancer and neuroprotective molecules. Out of the 120 experimentally validated CDPS members, only two are known to accept histidine as a substrate yielding cyclo(His-Phe) and cyclo(His-Pro) as products. It is not fully understood how CDPSs select their substrates, and we must rely on bioprospecting to find new enzymes and novel bioactive cyclic dipeptides. Here, we developed an in vitro system to generate an extensive library of molecules using canonical and non-canonical amino acids as substrates, expanding the chemical space of histidine-containing cyclic dipeptide analogues. To investigate substrate selection we determined the structure of a cyclo(His-Pro)-producing CDPS. Three consecutive generations harbouring single, double and triple residue substitutions elucidated the histidine selection mechanism. Moreover, substrate selection was redefined, yielding enzyme variants that became capable of utilising phenylalanine and leucine. Our work successfully engineered a CDPS to yield different products, paving the way to direct the promiscuity of these enzymes to produce molecules of our choosing.Publisher PDFPeer reviewe

Bypassing the requirement for aminoacyl-tRNA by a cyclodipeptide synthase enzyme

C. J. H. and C. M. C. are funded by the Wellcome trust (210486/Z/18/Z), ES is funded by the Cunningham trust (PhD-CT-18-41).Cyclodipeptide synthases (CDPSs) produce a variety of cyclic dipeptide products by utilising two aminoacylated tRNA substrates. We sought to investigate the minimal requirements for substrate usage in this class of enzymes as the relationship between CDPSs and their substrates remains elusive. Here, we investigated the Bacillus thermoamylovorans enzyme, BtCDPS, which synthesises cyclo(L-Leu–L-Leu). We systematically tested where specificity arises and, in the process, uncovered small molecules (activated amino esters) that will suffice as substrates, although catalytically poor. We solved the structure of BtCDPS to 1.7 Å and combining crystallography, enzymatic assays and substrate docking experiments propose a model for how the minimal substrates interact with the enzyme. This work is the first report of a CDPS enzyme utilizing a molecule other than aa-tRNA as a substrate; providing insights into substrate requirements and setting the stage for the design of improved simpler substrates.Publisher PDFPeer reviewe

Structure, dynamics, and molecular inhibition of the Staphylococcus aureus m1A22-tRNA methyltransferase TrmK

This work was supported by a Wellcome Trust Seed Award in Science [208980/Z/17/Z] to RGdS; a University of St Andrews/Scottish Funding Council St Andrews Restarting Research Fund to RGdS; and a Wellcome Trust Institutional Strategic Support Fund [204821/Z/16/Z] to the University of St Andrews. ES is the recipient of a Cunningham Trust PhD studentship (PhD-CT-18-41).The enzyme m1A22-tRNA methyltransferase (TrmK) catalyzes the transfer of a methyl group to the N1 of adenine 22 in bacterial tRNAs. TrmK is essential for Staphylococcus aureus survival during infection but has no homolog in mammals, making it a promising target for antibiotic development. Here, we characterize the structure and function of S. aureus TrmK (SaTrmK) using X-ray crystallography, binding assays, and molecular dynamics simulations. We report crystal structures for the SaTrmK apoenzyme as well as in complexes with methyl donor SAM and co-product product SAH. Isothermal titration calorimetry showed that SAM binds to the enzyme with favorable but modest enthalpic and entropic contributions, whereas SAH binding leads to an entropic penalty compensated for by a large favorable enthalpic contribution. Molecular dynamics simulations point to specific motions of the C-terminal domain being altered by SAM binding, which might have implications for tRNA recruitment. In addition, activity assays for SaTrmK-catalyzed methylation of A22 mutants of tRNALeu demonstrate that the adenine at position 22 is absolutely essential. In silico screening of compounds suggested the multifunctional organic toxin plumbagin as a potential inhibitor of TrmK, which was confirmed by activity measurements. Furthermore, LC-MS data indicated the protein was covalently modified by one equivalent of the inhibitor, and proteolytic digestion coupled with LC-MS identified Cys92 in the vicinity of the SAM-binding site as the sole residue modified. These results identify a cryptic binding pocket of SaTrmK, laying a foundation for future structure-based drug discovery.Publisher PDFPeer reviewe

Exploring biosynthetic routes to novel histidine containing cyclodipeptides

Abstract redacted"The research underpinning this thesis received funding from the Cunningham Trust (Grant: PhD-CT-18-41)."--Acknowledgement

Figure S3. LC-MS analysis of oxidised CDP substrates

LC-MS analysis to confirm oxidative products (CDP-ox1 and/or CDP-ox2) of CDPs by NdasCDO.</p

HERStory Makers 2022: Emmajay Sutherland

Emmajay Sutherland is a PhD candidate at the University of St Andrews studying development of anti-cancer drugs from environmental sources. She took part in HERStory Makers 2022.What is HERStory Makers?HERStory Makers is a social media competition for female-identifying early career researchers to share their research, their career journeys, and to inspire the next generation. Winners are selected by public vote. HERStory Makers is also part of EXPLORATHON, Scotland's contribution to European Researchers' Night.In 2022-23, EXPLORATHON was supported by the Engineering & Physical Sciences Research Council [grant number EP/X020762/1].Author contributions to contentEmmajay Sutherland conceived, planned, and recorded the video content. Kirsty Ross edited the video content to insert HERStory Maker credits, add subtitles, and maintain video length below Twitter/X limit of 2 mins and 20 secs, prior to scheduling the social media posts.</p

Figure 3d. LC-MS analysis of cFP oxidation over time

LC-MS analysis of cFP oxidation over time as catalysed by NdasCDO.</p

Active site remodelling of a cyclodipeptide synthase redefines substrate scope

Cyclodipeptide synthases (CDPSs) generate a wide range of cyclic dipeptides using aminoacylated tRNAs as substrates. Histidine-containing cyclic dipeptides have important biological activities as anticancer and neuroprotective molecules. Out of the 120 experimentally validated CDPS members, only two are known to accept histidine as a substrate yielding cyclo(His-Phe) and cyclo(His-Pro) as products. It is not fully understood how CDPSs select their substrates, and we must rely on bioprospecting to find new enzymes and novel bioactive cyclic dipeptides. Here, we developed an in vitro system to generate an extensive library of molecules using canonical and non-canonical amino acids as substrates, expanding the chemical space of histidine-containing cyclic dipeptide analogues. To investigate substrate selection we determined the structure of a cyclo(His-Pro)-producing CDPS. Three consecutive generations harbouring single, double and triple residue substitutions elucidated the histidine selection mechanism. Moreover, substrate selection was redefined, yielding enzyme variants that became capable of utilising phenylalanine and leucine. Our work successfully engineered a CDPS to yield different products, paving the way to direct the promiscuity of these enzymes to produce molecules of our choosing

Active site remodelling of a cyclodipeptide synthase redefines substrate scope

LC-MS data from paper entitled "Active site remodelling of a cyclodipeptide synthase redefines substrate scope

Erratum:Bypassing the requirement for aminoacyl-tRNA by a cyclodipeptide synthase enzyme (RSC Chem. Biol. (2021) 2 (230-240) DOI: 10.1039/D0CB00142B)

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