I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[39189]
Open Access
Abstract: The enzymes Cannabichromenic Acid Synthase (CBCAS), Cannabidiolic Acid Synthase (CBDAS) and Tetrahydrocannabinolic Acid Synthase (THCAS) are together the major cannabinoid synthase enzymes responsible for the biosynthesis of their respective metabolites from a common precursor Cannabigerolic Acid (CBGA). As the catalysts responsible for generating biological molecules of significant pharmaceutical value, there has been considerable interest in the enzymes with respect to heterologous production, mechanism, and incorporation into synthetic biology pathways for the facile industrial production of these molecules. The enzymes share high degrees of homology, and therefore their distinct specificities are governed by very subtle differences in sequence and therefore structure, although, until now, only a structure for THCAS has been reported. In this report, we present structures of CBCAS, CBDAS and a structure of THCAS at a higher resolution than the known structure, each in complex with their flavin coenzyme FAD. The structures reveal active site differences that may be responsible for the complementary activities observed, in terms of both first-shell amino acid substitutions, but also in more remote residues that influence active site topology through referred effects, or that have effects on substrate access. The structures provide a useful and informative platform for the rational engineering of improved or altered chemoselectivity in these enzymes.
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Dec 2026
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[32728]
Open Access
Abstract: Plant intracellular immune receptors are widely deployed in breeding to protect crops from disease. In addition to nucleotide-binding leucine-rich repeat receptors (NLRs), tandem kinase proteins (TKPs) have recently emerged as an important family of immune receptors within staple cereal food crops, but how TKPs recognize effectors and whether they are amenable to engineering is essentially unknown. Here, we show that the barley and wheat TKPs Rmo2 and Rwt7 recognize different blast fungus effectors via their integrated HMA domains using different protein interfaces with nanomolar binding affinity. Structural analysis pinpointed interface residues that dictate effector recognition and enabled engineering of dual-specificity TKPs. These results establish integrated HMA domains as programmable modules within TKPs for designing new specificities in plant immunity for diseases relevant to global agriculture.
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Sep 2026
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[28402, 35088]
Open Access
Abstract: Keratin is an abundant structural fibrous protein and extremely recalcitrant biopolymer. β-Keratin is the major constituent of feathers, which, due to the widespread poultry industry, has become a major waste product. Biotechnological upcycling of feather waste has gained interest as various bacteria and fungi capable of degrading keratin have been isolated. These microorganisms produce proteases, termed keratinases, responsible for the enzymatic hydrolysis of keratin. The structural properties that confer keratinolytic activity to proteases are, however, not well understood. Here, we investigated the structure-function relationship of a subtilisin-like S8 endopeptidase (FerB) from the thermophile Fervidobacterium pennivorans strain T. FerB was crystallized and its structure solved to 1.5 Å resolution, revealing an auto-processed state where the pro-peptide domain is non-covalently attached to the catalytic domain. The carboxyl group of the scissile peptide bond is coordinated in the active site within hydrogen bonding distance of the catalytic triad’s serine residue. Unlike fervidolysin, no β-sandwich domains are present. However, a tyrosine-rich β-hairpin structure is found in the corresponding position within the FerB structure. Deletion of the β-hairpin reduced the protein’s integrity and keratinase activity.
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Aug 2026
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[39189]
Open Access
Abstract: ATP-grasp enzymes, such as l-amino acid ligases (Lals), catalyze the coupling of carboxylate and amino acid acyl donor substrates to amino acid acyl acceptor substrates via an acyl phosphate intermediate. In addition to their roles in the biosynthesis of natural products, ATP-grasp enzymes have generated significant interest as preparative biocatalysts for the selective synthesis of peptides and amides. TabS is a Lal from Pseudomonas syringae NBRC14081 that couples l-threonine and tabtoxinine-β-lactam to give tabtoxin, which, when hydrolyzed, yields tabtoxinine-β-lactam as a glutamine synthetase inhibitor. However, TabS also has a broad substrate specificity encompassing the coupling of many pairs of l-amino acids, including l-leucine and l-phenylalanine, to form a range of homo- and heterodipeptides. Here we report the structure of TabS, in an apo- form and also in complex with the noncleavable ATP analog AMP-PNP, refined to resolutions of 2.17 and 2.90 Å, respectively. The structures shed light on the broad substrate specificity of TabS and have also informed the exploration, through modeling, mutation, and activity assays, of the possibilities of engineering these enzymes for the coupling of simple carboxylates and amines to form nonpeptide amide products. Rational mutation of hydrophilic residues thought to recognize the amino group of acyl donors and the carboxylate of acyl acceptors has given variants, including S16A/Y44F/Y45F/Y235F/L382F/Q337L/Q339L, which successfully convert the non-amino-acid analogs of the l-leucine donor and acceptor, i.e., 4-methyl pentanoate and 3-methyl butylamine, respectively, to the nonpeptide amide product, where the wild-type TabS displayed negligible activity.
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Aug 2026
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Laura N.
Jeffreys
,
Sian
Thistlethwaite
,
Matthew J.
Cliff
,
Harshwardhan
Poddar
,
Sahara
Bhanot
,
Richard B.
Tunnicliffe
,
Marina
Golovanova
,
Katherine
Hollywood
,
Colin W.
Levy
,
Michael W.
Voice
,
David
Leys
,
Kirsty J.
Mclean
,
Jonathan P.
Waltho
,
Andrew W.
Munro
,
Hazel M.
Girvan
Diamond Proposal Number(s):
[17773, 24447]
Open Access
Abstract: P450 BM3 is a natural fusion protein with a P450 domain fused to its necessary redox partner and capable of binding a range of fatty acids at high catalytic rates. The flexibility of the active site and catalytic efficiency have led to extensive research aimed at modifying substrate and product profiles for its utilization as a biocatalyst. The introduction of a double mutation (DM) at the base of the active site (A82F/F87V) allows the binding of a broad range of structurally diverse small molecules, including pharmaceutical drugs. Herein, we describe screening with an FDA-approved drug compound library, and the results exhibit the promiscuous nature of the DM BM3 variant. Of the 978 compounds screened; 59% of the library elicited UV–vis spectral shifts indicative of binding with a range of structurally diverse drugs. The interaction of the DM BM3 variant with a range of structurally diverse drugs, with a particular focus on the antidiabetic glitazone (thiazolidinedione) class, was investigated to observe ligand binding modes and metabolite production. BM3-derived metabolites were successfully produced and structurally elucidated for several structurally diverse drugs in the steroid, glitazone, meglitinide, terpene lactone, retinoid, and fibrate classes. The range of modifications performed includes some that are human P450-derived metabolites. Structural elucidation of the troglitazone-bound DM variant P450 domain shows an unusually close crystallographic interaction between the substrate and heme cofactor. We report here the use of the DM BM3 variant as a model for the main human metabolizing P450 enzymes CYP1A1, CYP2B6, CYP2C8, CYP2C9, and CYP3A4, as well as a biotechnological tool for the production of metabolites with diverse functionality.
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Aug 2026
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Massimo
Tedeschi
,
Vincent A. J.
Lit
,
Nicholas G. S.
Mcgregor
,
Tessa
Gote
,
Prajeesh
Kooloth Valappil
,
Mark
Arentshorst
,
Bogdan I.
Florea
,
Berend
Gagestein
,
Zachary
Armstrong
,
Jeroen D. C.
Codée
,
Alba
Nin-Hill
,
Carme
Rovira
,
Arthur F. J.
Ram
,
Gideon J.
Davies
,
Herman S.
Overkleeft
Diamond Proposal Number(s):
[24948]
Open Access
Abstract: β-Mannanases are endo-acting glycoside hydrolases (GHs) that cleave β-1,4 glycosidic linkages in mannan-rich plant cell wall polysaccharides. They find application in the food and paper industries. Activity-based probes (ABPs) are powerful tools for GH profiling in complex biological samples, yet to date, bespoke ABPs reporting on mannanases have not been reported. Here, we describe the synthesis of cyclophellitol-inspired ABPs based on mannobiose, mannotriose, and glucomannose, and their use in reporting mannanase activities in secretomes from saprophytic bacteria and fungi grown on mannan-containing biomass polysaccharides. In addition to mannanases, our ABPs also labelled cellulases in secretomes from both Aspergillus niger and Cellvibrio japonicus, which may indicate broader (“negative-subsite”) substrate specificity in these enzymes. Mechanistic proof of active-site nucleophile labelling by our ABPs was obtained for both AnManA and CjMan26C by X-ray crystallography and for both AnManA and AnMan26A by mass spectrometry. Together, our results establish mannanase-targeted ABPs that may find use alongside existing reagents that report on retaining GHs that process other bulk polysaccharides.
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Jul 2026
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23269]
Abstract: Lignin is a sustainable alternative to petroleum as a feedstock for the chemical industry. Emergent strategies for lignin valorization involve tandem processes in which biomass is chemo-catalytically fractionated, followed by bioconversion of the depolymerized lignin by microbial cell factories. A rate-limiting step in this bioconversion is O-demethylation of the lignin-derived monomers. The reductive catalytic fractionation of hardwood biomass generates high yields of two classes of monomers: 4-alkylguaiacols and 4-alkylsyringols. The former are O-demethylated by AgcA, a cytochrome P450, and AgcB, the cognate reductase, but there are no known enzymes that convert the latter. To develop a biocatalyst that can efficiently transform these monomers, we studied and rationally engineered AgcAB. A 1.82 Å resolution crystal structure of AgcAEP4 from Rhodococcus rhodochrous EP4 in complex with 4-ethylguaiacol identified residues Leu78, Ala293, and Phe166 as potential specificity determinants. Substitution of Ala293 and Leu78 decreased the specificity of AgcAEP4 for alkylguaiacols. Substitution of Phe166 yielded a variant that bound 4-propylsyringol but did not transform it. In contrast, the corresponding variant in the Rhodococcus aromaticivorans RHA1 homologue, AgcARHA1 Y166A, catalyzed the O-demethylation of both methoxy groups of 4-propylsyringol with a kcat/Km of 8500 M–1 s–1 for the first O-demethylation, nearly 7-fold higher than WT AgcARHA1. Engineering RHA1 to express the variant yielded a strain that transformed 4-propylsyringol and 4-propylguaiacol simultaneously. Moreover, the engineered strain converted some of the 4-propylsyringol to pentanoyl-CoA, consistent with catabolism via the meta-cleavage pathway that catabolizes 4-alkylguaiacols. Exometabolomics validated the conversion of 4-propylsyringol via this pathway and identified O-demethylation and extradiol ring cleavage as bottlenecks for its transformation. These studies improve our understanding of a critical lignin-degrading enzyme system and significantly advance the development of a biocatalyst to convert these monomers.
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Jul 2026
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[38144]
Open Access
Abstract: [FeFe]-hydrogenases are the fastest enzymes for hydrogen evolution, yet their irreversible inhibition by oxygen has thwarted their industrial use. CbA5H is an exception as its inhibition by oxygen is reversible. Protein scaffold rearrangement near the active site, allowing a ligand to coordinate the metal center in addition to the formation of a highly oxidized state of the metal center named Hinact, is the current hypothesis for CbA5H oxygen stability. However, the ligand identity has been disputed and there is no evidence to suggest that protein scaffold rearrangement is the sole reason for oxygen stability. Here, we investigate CbA5H oxygen stability by providing a high-resolution (1.96 Å) X-ray structure that shows that the protective ligand is a conserved cysteine thiol group, which directly coordinates the metal center. The local rearrangement also encompasses structural water molecules and the side chain of E341, associated with proton transfer. In addition, we illustrate that C236 and H245, located close to accessory iron sulfur clusters in the Fd domain, influence oxygen stability. We show that mutating these residues significantly decreases oxygen stability but not the ability to form Hinact. Variant C236A displays a slower inactivation rate, which we suggest is due to tuning the redox properties of one of the accessory iron sulfur clusters. We also show that the soluble ligand-binding β-grasp domain (SLBB) may not be required for oxygen stability by comparing CbA5H to a novel homolog lacking this domain. Collectively, these findings expand our understanding of oxygen stability in [FeFe]-hydrogenases.
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Jul 2026
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I24-Microfocus Macromolecular Crystallography
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Simon
D'Oelsnitz
,
Wantae
Kim
,
Nicole N.
Zhao
,
Haley
Hardtke
,
Svetlana P.
Ikonomova
,
Nina
Alperovich
,
Olga
Vasilyeva
,
Michael J.
James
,
Eric S.
Zigon
,
Michael B.
Cory
,
Charlie D.
Johnson
,
Andrew D.
Ellington
,
Quincey A.
Justman
,
Michael
Springer
,
Y. Jessie
Zhang
,
Pamela A.
Silver
,
David
Ross
Diamond Proposal Number(s):
[36008]
Open Access
Abstract: Biocatalysts are prized for their enantioselectivity, but slow chromatographic separations required to measure enantiomeric excess bottleneck their development. To overcome this limitation, we evolve enantioselective transcription factors (eTFs) that convert enzyme-catalyzed enantiomer concentrations into programmable gene expression outputs, focusing on imine reductases. Here, using a massively parallel reporter assay, we measure dose–response curves for over 300,000 transcription factor variants in response to an imine precursor and chiral amine products. We quantify the sensitivity, selectivity and dynamic range across variants generated by random, site-saturation and shuffling mutagenesis, isolating variants with exceptional specificity. High-resolution structures of evolved eTFs elucidate how steric effects enforce enantioselectivity, while charge interactions distinguish the imine from the amines. Using two eTFs, we create an ultrahigh-throughput chiral screen to evolve an imine reductase with inverted enantioselectivity. To support generalizability and speed, we design a genetic circuit that enables TF generation within weeks. Our methods enable rapid measurement of asymmetric reactions, supporting innovation in chemical manufacturing.
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Jul 2026
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I24-Microfocus Macromolecular Crystallography
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Abstract: The bacterial cytochrome P450 peroxygenase family (CYP152) represents a unique class of heme-containing monooxygenases that utilise hydrogen peroxide as an oxidant, bypassing the canonical P450 redox partner system. Members of this family, particularly OleTJE, originally isolated from Jeotgalicoccus sp. ATCC 8456, are capable of catalysing oxidative decarboxylation of fatty acids to produce terminal alkenes (olefins). These olefins are of growing industrial interest as potential drop-in biofuels. The key reactive intermediate in this catalytic cycle is the ferryl-oxo porphyrin π-cation radical, commonly referred to as Compound I. However, due to its high reactivity and transient nature, direct characterisation of Compound I remains a significant challenge. This study focuses on engineering the hydrogen-bonding network surrounding the heme-b prosthetic group in OleTJE through site-directed mutagenesis, with the aim of extending the lifetime of Compound I. A combination of structural, kinetic, and spectroscopic techniques was employed to characterise the impact of these mutations. Fatty acid binding titrations revealed altered binding affinities through dissociation constants, stopped-flow UV/Vis spectrophotometry indicated a significant reduction in Compound I decay rate, and high-resolution macromolecular crystallography provided structural insights into the modified active site architecture. Future studies will incorporate techniques such as electron paramagnetic resonance (EPR) spectroscopy to investigate changes in the heme iron spin state equilibrium, offering deeper mechanistic insight into how these mutations influence the electronic environment of the active site as well as GC-MS analysis to confirm product formation and fatty acid presence post-purification. Collectively, these results demonstrate the potential of rational protein engineering to stabilise reactive intermediates and enhance enzymatic performance for biotechnological applications.
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Jun 2026
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