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Title: Crystallographic and kinetic study of riboflavin synthase from Brucella abortus, a chemotherapeutic target with an enhanced intrinsic flexibility

This work reports crystal structures of trimeric riboflavin synthase from the pathogen B. abortus both as the apo protein and in complex with several ligands of interest. It is shown that ligand binding drives the assembly of the unique active site of the trimer, and these findings are complemented by a detailed kinetic study on this enzyme, in which marked inhibition by substrate and product was observed. Riboflavin synthase (RS) catalyzes the last step of riboflavin biosynthesis in microorganisms and plants, which corresponds to the dismutation of two molecules of 6,7-dimethyl-8-ribityllumazine to yield one molecule of riboflavin and one molecule of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. Owing to the absence of this enzyme in animals and the fact that most pathogenic bacteria show a strict dependence on riboflavin biosynthesis, RS has been proposed as a potential target for antimicrobial drug development. Eubacterial, fungal and plant RSs assemble as homotrimers lacking C{sub 3} symmetry. Each monomer can bind two substrate molecules, yet there is only one active site for the whole enzyme, which is located at the interface between two neighbouring chains. This work reports the crystallographic structure of RS from the pathogenic bacterium Brucella abortus (the aetiological agent of the disease brucellosis) in itsmore » apo form, in complex with riboflavin and in complex with two different product analogues, being the first time that the structure of an intact RS trimer with bound ligands has been solved. These crystal models support the hypothesis of enhanced flexibility in the particle and also highlight the role of the ligands in assembling the unique active site. Kinetic and binding studies were also performed to complement these findings. The structural and biochemical information generated may be useful for the rational design of novel RS inhibitors with antimicrobial activity.« less
Authors:
;  [1] ;  [2] ;  [3] ; ;  [1]
  1. IIBBA–CONICET, Avenida Patricias Argentinas 435, C1405BWE Buenos Aires (Argentina)
  2. Synchrotron SOLEIL, L’Orme des Merisiers, Saint-Aubin BP 48, 91192 Gif-sur-Yvette CEDEX (France)
  3. Universidad de Buenos Aires, Junín 956, C1113AAD Buenos Aires (Argentina)
Publication Date:
OSTI Identifier:
22351319
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section D: Biological Crystallography; Journal Volume: 70; Journal Issue: Pt 5; Other Information: PMCID: PMC4014124; PMID: 24816110; PUBLISHER-ID: kw5091; OAI: oai:pubmedcentral.nih.gov:4014124; Copyright (c) International Union of Crystallography 2014; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
Denmark
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; CHAINS; CRYSTAL MODELS; CRYSTAL STRUCTURE; DESIGN; FLEXIBILITY; INTERFACES; LIGANDS; MOLECULES; MONOMERS; POTENTIALS; SUBSTRATES; SYMMETRY; YIELDS