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Instruments / Technical Area	Publication Details	Published
I04-1-Macromolecular Crystallography (fixed wavelength)	d-Cycloserine destruction by alanine racemase and the limit of irreversible inhibition Cesira De Chiara , Miha Homšak , Gareth A. Prosser , Holly L. Douglas , Acely Garza-garcia , Geoff Kelly , Andrew G. Purkiss , Edward W. Tate , Luiz Pedro S. De Carvalho Nature Chemical Biology 5 DOI: 10.1038/s41589-020-0498-9 Diamond Proposal Number(s): [13775] Show Abstract ▼ Abstract: The broad-spectrum antibiotic D-cycloserine (DCS) is a key component of regimens used to treat multi- and extensively drug-resistant tuberculosis. DCS, a structural analog of D-alanine, binds to and inactivates two essential enzymes involved in peptidoglycan biosynthesis, alanine racemase (Alr) and D-Ala:D-Ala ligase. Inactivation of Alr is thought to proceed via a mechanism-based irreversible route, forming an adduct with the pyridoxal 5′-phosphate cofactor, leading to bacterial death. Inconsistent with this hypothesis, Mycobacterium tuberculosis Alr activity can be detected after exposure to clinically relevant DCS concentrations. To address this paradox, we investigated the chemical mechanism of Alr inhibition by DCS. Inhibition of M. tuberculosis Alr and other Alrs is reversible, mechanistically revealed by a previously unidentified DCS-adduct hydrolysis. Dissociation and subsequent rearrangement to a stable substituted oxime explains Alr reactivation in the cellular milieu. This knowledge provides a novel route for discovery of improved Alr inhibitors against M. tuberculosis and other bacteria.	Mar 2020
I04-Macromolecular Crystallography	Uncoupling conformational states from activity in an allosteric enzyme Joao P. Pisco , Cesira De Chiara , Kamila J. Pacholarz , Acely Garza-garcia , Roksana W. Ogrodowicz , Philip A. Walker , Perdita E. Barran , Stephen J. Smerdon , Luiz Pedro S. De Carvalho Nature Communications 8 DOI: 10.1038/s41467-017-00224-0 Diamond Proposal Number(s): [7707, 9826] Open Access Show Abstract ▼ Abstract: ATP-phosphoribosyltransferase (ATP-PRT) is a hexameric enzyme in conformational equilibrium between an open and seemingly active state and a closed and presumably inhibited form. The structure-function relationship of allosteric regulation in this system is still not fully understood. Here, we develop a screening strategy for modulators of ATP-PRT and identify 3-(2-thienyl)-l-alanine (TIH) as an allosteric activator of this enzyme. Kinetic analysis reveals co-occupancy of the allosteric sites by TIH and l-histidine. Crystallographic and native ion-mobility mass spectrometry data show that the TIH-bound activated form of the enzyme closely resembles the inhibited l-histidine-bound closed conformation, revealing the uncoupling between ATP-PRT open and closed conformations and its functional state. These findings suggest that dynamic processes are responsible for ATP-PRT allosteric regulation and that similar mechanisms might also be found in other enzymes bearing a ferredoxin-like allosteric domain.	Aug 2017
	Characterization of Vibrio cholerae Hfq Provides Novel Insights into the Role of the Hfq C-Terminal Region Helen A. Vincent , Charlotte Henderson , Timothy J. Ragan , Acely Garza-garcia , Peter D. Cary , Darren Gowers , Marc Malfois , Paul C. Driscoll , Frank Sobott , Anastasia Callaghan Journal Of Molecular Biology 420, 56 - 69 DOI: 10.1016/j.jmb.2012.03.028 PMID: 22484176 Open Access Show Abstract ▼ Abstract: Hfq is a bacterial RNA binding protein that facilitates small RNA-mediated posttranscriptional gene regulation. In Vibrio cholerae, Hfq and four Hfq-dependent small RNAs are essential for the expression of virulence genes, but little is known about this mechanism at the molecular level. To better understand V. cholerae Hfq structure and mechanism, we characterized the protein, alongside Escherichia coli Hfq for comparison, using biochemical and biophysical techniques. The N-terminal domain (NTD) of the two proteins is highly conserved, but the C-terminal regions (CTRs) vary in both sequence and length. Small-angle X-ray scattering studies showed that both proteins adopt a star-shaped hexameric structure in which the conserved NTD adopts the expected Sm fold while the variable CTR is disordered and extends radially outwards from the folded core. Despite their structural similarity, SDS-PAGE stability assays and collision-induced dissociation mass spectrometry revealed that the V. cholerae hexamer is less stable than that of E. coli. We propose that this is due to minor differences between the intersubunit interface formed by the NTDs and the ability of the E. coli CTR to stabilize this interface. However, based on electrophoretic mobility shift assays, the divergent CTRs do appear to perform a common function with regard to RNA-binding specificity. Overall, the similarities and differences in the fundamental properties of V. cholerae and E. coli Hfq provide insight into their assembly and molecular mechanisms.	Jun 2012

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