Krios I-Titan Krios I at Diamond
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Open Access
Abstract: Cyclic dipeptides are produced by organisms across all domains of life, with many exhibiting anticancer and antimicrobial properties. Oxidations are often key to their biological activities, particularly C-C bond oxidation catalysed by tailoring enzymes including cyclodipeptide oxidases. These flavin-dependent enzymes are underexplored due to their intricate three-dimensional arrangement involving multiple copies of two distinct small subunits, and mechanistic details underlying substrate selection and catalysis are lacking. Here, we determined the structure and mechanism of the cyclodipeptide oxidase from the halophile Nocardiopsis dassonvillei (NdasCDO), a component of the biosynthetic pathway for nocazine natural products. We demonstrated that NdasCDO forms filaments in solution, with a covalently bound flavin mononucleotide (FMN) cofactor at the interface between three distinct subunits. The enzyme exhibits promiscuity, processing various cyclic dipeptides as substrates in a distributive manner. The reaction is optimal at high pH and involves the formation of a radical intermediate. Pre-steady-state kinetics, a significant solvent kinetic isotope effect, and the absence of viscosity effects suggested that a step linked to FMN regeneration controlled the reaction rate. Our work elucidates the complex mechanistic and structural characteristics of this dehydrogenation reaction, positioning NdasCDO as a promising biocatalyst and expanding the FMN-dependent oxidase family to include enzyme filaments.
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Jan 2025
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[26793, 19844]
Open Access
Abstract: Nucleosides are ubiquitous to life and are required for the synthesis of DNA, RNA, and other molecules crucial for cell survival. Despite the notoriously difficult organic synthesis of nucleosides, 2′-deoxynucleoside analogues can interfere with natural DNA replication and repair and are successfully employed as anticancer, antiviral, and antimicrobial compounds. Nucleoside 2′-deoxyribosyltransferase (dNDT) enzymes catalyze transglycosylation via a covalent 2′-deoxyribosylated enzyme intermediate with retention of configuration, having applications in the biocatalytic synthesis of 2′-deoxynucleoside analogues in a single step. Here, we characterize the structure and function of a thermophilic dNDT, the protein from Chroococcidiopsis thermalis (CtNDT). We combined enzyme kinetics with structural and biophysical studies to dissect mechanistic features in the reaction coordinate, leading to product formation. Bell-shaped pH-rate profiles demonstrate activity in a broad pH range of 5.5–9.5, with two very distinct pKa values. A pronounced viscosity effect on the turnover rate indicates a diffusional step, likely product (nucleobase1) release, to be rate-limiting. Temperature studies revealed an extremely curved profile, suggesting a large negative activation heat capacity. We trapped a 2′-fluoro-2′-deoxyarabinosyl-enzyme intermediate by mass spectrometry and determined high-resolution structures of the protein in its unliganded, substrate-bound, ribosylated, 2′-difluoro-2′-deoxyribosylated, and in complex with probable transition-state analogues. We reveal key features underlying (2′-deoxy)ribonucleoside selection, as CtNDT can also use ribonucleosides as substrates, albeit with a lower efficiency. Ribonucleosides are the building blocks of RNA and other key intracellular metabolites participating in energy and metabolism, expanding the scope of use of CtNDT in biocatalysis.
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Feb 2024
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[26793]
Open Access
Abstract: Intracellular leucine aminopeptidases (PepA) are metalloproteases from the family M17. These enzymes catalyze peptide bond cleavage, removing N-terminal residues from peptide and protein substrates, with consequences for protein homeostasis and quality control. While general mechanistic studies using model substrates have been conducted on PepA enzymes from various organisms, specific information about their substrate preferences and promiscuity, choice of metal, activation mechanisms, and the steps that limit steady-state turnover remain unexplored. Here, we dissected the catalytic and chemical mechanisms of PaPepA: a leucine aminopeptidase from Pseudomonas aeruginosa. Cleavage assays using peptides and small-molecule substrate mimics allowed us to propose a mechanism for catalysis. Steady-state and pre-steady-state kinetics, pH rate profiles, solvent kinetic isotope effects, and biophysical techniques were used to evaluate metal binding and activation. This revealed that metal binding to a tight affinity site is insufficient for enzyme activity; binding to a weaker affinity site is essential for catalysis. Progress curves for peptide hydrolysis and crystal structures of free and inhibitor-bound PaPepA revealed that PaPepA cleaves peptide substrates in a processive manner. We propose three distinct modes for activity regulation: tight packing of PaPepA in a hexameric assembly controls substrate length and reaction processivity; the product leucine acts as an inhibitor, and the high concentration of metal ions required for activation limits catalytic turnover. Our work uncovers catalysis by a metalloaminopeptidase, revealing the intricacies of metal activation and substrate selection. This will pave the way for a deeper understanding of metalloenzymes and processive peptidases/proteases.
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Nov 2023
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[26793]
Open Access
Abstract: Pseudomonas aeruginosa is an opportunistic pathogen that causes serious illness, especially in immunocompromised individuals. P. aeruginosa forms biofilms that contribute to growth and persistence in a wide range of environments. Here we investigated the aminopeptidase, P. aeruginosa aminopeptidase (PaAP) from P. aeruginosa, which is highly abundant in the biofilm matrix. PaAP is associated with biofilm development and contributes to nutrient recycling. We confirmed that post-translational processing was required for activation and PaAP is a promiscuous aminopeptidase acting on unstructured regions of peptides and proteins. Crystal structures of wild-type enzymes and variants revealed the mechanism of autoinhibition, whereby the C-terminal propeptide locks the protease-associated domain and the catalytic peptidase domain into a self-inhibited conformation. Inspired by this, we designed a highly potent small cyclic-peptide inhibitor that recapitulates the deleterious phenotype observed with a PaAP deletion variant in biofilm assays and present a path toward targeting secreted proteins in a biofilm context.
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Jun 2023
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[26793]
Open Access
Abstract: 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.
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Aug 2022
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14692]
Open Access
Abstract: Bacterial hybrid malic enzymes (MaeB grouping, multidomain) catalyse the transformation of malate to pyruvate, and are a major contributor to cellular reducing power and carbon flux. Distinct from other malic enzyme subtypes, the hybrid enzymes are regulated by acetyl-CoA, a molecular indicator of the metabolic state of the cell. Here we solve the structure of a MaeB protein, which reveals hybrid enzymes use the appended phosphotransacetylase (PTA) domain to form a hexameric sensor that communicates acetyl-CoA occupancy to the malic enzyme active site, 60 Å away. We demonstrate that allostery is governed by a large-scale rearrangement that rotates the catalytic subunits 70° between the two states, identifying MaeB as a new model enzyme for the study of ligand-induced conformational change. Our work provides the mechanistic basis for metabolic control of hybrid malic enzymes, and identifies inhibition-insensitive variants that may find utility in synthetic biology.
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Feb 2021
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19844]
Open Access
Abstract: 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.
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Jan 2021
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14692, 19880]
Open Access
Abstract: Lysozymes are among the best-characterized enzymes, acting upon the cell wall substrate peptidoglycan. Here, examining the invasive bacterial periplasmic predator Bdellovibrio bacteriovorus, we report a diversified lysozyme, DslA, which acts, unusually, upon (GlcNAc-) deacetylated peptidoglycan. B. bacteriovorus are known to deacetylate the peptidoglycan of the prey bacterium, generating an important chemical difference between prey and self walls and implying usage of a putative deacetyl-specific “exit enzyme”. DslA performs this role, and ΔDslA strains exhibit a delay in leaving from prey. The structure of DslA reveals a modified lysozyme superfamily fold, with several adaptations. Biochemical assays confirm DslA specificity for deacetylated cell wall, and usage of two glutamate residues for catalysis. Exogenous DslA, added ex vivo, is able to prematurely liberate B. bacteriovorus from prey, part-way through the predatory lifecycle. We define a mechanism for specificity that invokes steric selection, and use the resultant motif to identify wider DslA homologues.
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Sep 2020
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Rosalba
Lepore
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Andriy
Kryshtafovych
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Markus
Alahuhta
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Harshul A.
Veraszto
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Yannick J.
Bomble
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Joshua C.
Bufton
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Alex N.
Bullock
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Cody
Caba
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Hongnan
Cao
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Owen R.
Davies
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Ambroise
Desfosses
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Matthew
Dunne
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Krzysztof
Fidelis
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Celia W.
Goulding
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Manickam
Gurusaran
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Irina
Gutsche
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Christopher J.
Harding
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Marcus D.
Hartmann
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Christopher S.
Hayes
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Andrzej
Joachimiak
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Petr G.
Leiman
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Peter
Loppnau
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Andrew L.
Lovering
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Vladimir V.
Lunin
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Karolina
Michalska
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Ignacio
Mir‐sanchis
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Alok
Mitra
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John
Moult
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George N.
Phillips Jr
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Daniel
Pinkas
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Phoebe A.
Rice
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Yufeng
Tong
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Maya
Topf
,
Jonathan D.
Walton
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Torsten
Schwede
Diamond Proposal Number(s):
[14692]
Open Access
Abstract: The functional and biological significance of selected CASP13 targets are described by the authors of the structures. The structural biologists discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP13 experiment.
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Sep 2019
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I04-Macromolecular Crystallography
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Gareth W.
Hughes
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Stephen C. L.
Hall
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Claire S.
Laxton
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Pooja
Sridhar
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Amirul H.
Mahadi
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Caitlin
Hatton
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Thomas J.
Piggot
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Peter J.
Wotherspoon
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Aneika C.
Leney
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Douglas G.
Ward
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Mohammed
Jamshad
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Vaclav
Spana
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Ian T.
Cadby
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Christopher
Harding
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Georgia L.
Isom
,
Jack A.
Bryant
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Rebecca J.
Parr
,
Yasin
Yakub
,
Mark
Jeeves
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Damon
Huber
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Ian R.
Henderson
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Luke A.
Clifton
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Andrew L.
Lovering
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Timothy J.
Knowles
Diamond Proposal Number(s):
[14692]
Abstract: The Mla pathway is believed to be involved in maintaining the asymmetrical Gram-negative outer membrane via retrograde phospholipid transport. The pathway is composed of three components: the outer membrane MlaA–OmpC/F complex, a soluble periplasmic protein, MlaC, and the inner membrane ATPase, MlaFEDB complex. Here, we solve the crystal structure of MlaC in its phospholipid-free closed apo conformation, revealing a pivoting β-sheet mechanism that functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC, we provide evidence that the inner-membrane MlaFEDB machinery exports phospholipids to MlaC in the periplasm. Furthermore, we confirm that the phospholipid export process occurs through the MlaD component of the MlaFEDB complex and that this process is independent of ATP. Our data provide evidence of an apparatus for lipid export away from the inner membrane and suggest that the Mla pathway may have a role in anterograde phospholipid transport.
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Jun 2019
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