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      Predicting Protein Function from Structure—The Roles of Short-chain Dehydrogenase/Reductase Enzymes in Bordetella O-antigen Biosynthesis

      research-article
      1 , 2 , * , , 2 , 2 , , 2 , 1 , 3 , 2 , 2 , 4 , 2 , 4 , 2 , 2 , 1
      Journal of Molecular Biology
      Elsevier
      dTDP, deoxythymidine diphosphate, l-GalNAc3NAcA, 2,3-diacetamido-2,3-dideoxy-l-galacturonic acid, GME, GDP-mannose 3,5-epimerase, GMER, GDP-4-keto-6-deoxymannose 3,5-epimerase/reductase, LPS, lipopolysaccharide, PDB, Protein Data Bank, BLAST, basic local alignment search tool, SDR, short-chain dehydrogenase/reductase, UDP-d-ManNAc3NAcA, UDP-2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, UDP-l-GalNAc3NAcA, UDP-2,3-diacetamido-2,3-dideoxy-l-galacturonic acid, UMP, uridine monophosphate, MCS, multiple cloning site, short-chain dehydrogenase/reductase, X-ray crystallography, Bordetella, lipopolysaccharide, O-antigen biosynthesis

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          Abstract

          The pathogenic bacteria Bordetella parapertussis and Bordetella bronchiseptica express a lipopolysaccharide O antigen containing a polymer of 2,3-diacetamido-2,3-dideoxy- l-galacturonic acid. The O-antigen cluster contains three neighbouring genes that encode proteins belonging to the short-chain dehydrogenase/reductase (SDR) family, wbmF, wbmG and wbmH, and we aimed to elucidate their individual functions. Mutation and complementation implicate each gene in O-antigen expression but, as their putative sugar nucleotide substrates are not currently available, biochemical characterisation of WbmF, WbmG and WbmH is impractical at the present time. SDR family members catalyse a wide range of chemical reactions including oxidation, reduction and epimerisation. Because they typically share low sequence conservation, however, catalytic function cannot be predicted from sequence analysis alone. In this context, structural characterisation of the native proteins, co-crystals and small-molecule soaks enables differentiation of the functions of WbmF, WbmG and WbmH. These proteins exhibit typical SDR architecture and coordinate NAD. In the substrate-binding domain, all three enzymes bind uridyl nucleotides. WbmG contains a typical SDR catalytic TYK triad, which is required for oxidoreductase function, but the active site is devoid of additional acid–base functionality. Similarly, WbmH possesses a TYK triad, but an otherwise feature-poor active site. Consequently, 3,5-epimerase function can probably be ruled out for these enzymes. The WbmF active site contains conserved 3,5-epimerase features, namely, a positionally conserved cysteine (Cys133) and basic side chain (His90 or Asn213), but lacks the serine/threonine component of the SDR triad and therefore may not act as an oxidoreductase. The data suggest a pathway for synthesis of the O-antigen precursor UDP-2,3-diacetamido-2,3-dideoxy- l-galacturonic acid and illustrate the usefulness of structural data in predicting protein function.

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          Structure validation by Calpha geometry: phi,psi and Cbeta deviation.

          Geometrical validation around the Calpha is described, with a new Cbeta measure and updated Ramachandran plot. Deviation of the observed Cbeta atom from ideal position provides a single measure encapsulating the major structure-validation information contained in bond angle distortions. Cbeta deviation is sensitive to incompatibilities between sidechain and backbone caused by misfit conformations or inappropriate refinement restraints. A new phi,psi plot using density-dependent smoothing for 81,234 non-Gly, non-Pro, and non-prePro residues with B < 30 from 500 high-resolution proteins shows sharp boundaries at critical edges and clear delineation between large empty areas and regions that are allowed but disfavored. One such region is the gamma-turn conformation near +75 degrees,-60 degrees, counted as forbidden by common structure-validation programs; however, it occurs in well-ordered parts of good structures, it is overrepresented near functional sites, and strain is partly compensated by the gamma-turn H-bond. Favored and allowed phi,psi regions are also defined for Pro, pre-Pro, and Gly (important because Gly phi,psi angles are more permissive but less accurately determined). Details of these accurate empirical distributions are poorly predicted by previous theoretical calculations, including a region left of alpha-helix, which rates as favorable in energy yet rarely occurs. A proposed factor explaining this discrepancy is that crowding of the two-peptide NHs permits donating only a single H-bond. New calculations by Hu et al. [Proteins 2002 (this issue)] for Ala and Gly dipeptides, using mixed quantum mechanics and molecular mechanics, fit our nonrepetitive data in excellent detail. To run our geometrical evaluations on a user-uploaded file, see MOLPROBITY (http://kinemage.biochem.duke.edu) or RAMPAGE (http://www-cryst.bioc.cam.ac.uk/rampage). Copyright 2003 Wiley-Liss, Inc.
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            A graphical user interface to the CCP4 program suite.

            CCP4i is a graphical user interface that makes running programs from the CCP4 suite simpler and quicker. It is particularly directed at inexperienced users and tightly linked to introductory and scientific documentation. It also provides a simple project-management system and visualization tools. The system is readily extensible and not specific to CCP4 software.
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              Comparative analysis of the genome sequences of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica.

              Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica are closely related Gram-negative beta-proteobacteria that colonize the respiratory tracts of mammals. B. pertussis is a strict human pathogen of recent evolutionary origin and is the primary etiologic agent of whooping cough. B. parapertussis can also cause whooping cough, and B. bronchiseptica causes chronic respiratory infections in a wide range of animals. We sequenced the genomes of B. bronchiseptica RB50 (5,338,400 bp; 5,007 predicted genes), B. parapertussis 12822 (4,773,551 bp; 4,404 genes) and B. pertussis Tohama I (4,086,186 bp; 3,816 genes). Our analysis indicates that B. parapertussis and B. pertussis are independent derivatives of B. bronchiseptica-like ancestors. During the evolution of these two host-restricted species there was large-scale gene loss and inactivation; host adaptation seems to be a consequence of loss, not gain, of function, and differences in virulence may be related to loss of regulatory or control functions.
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                Author and article information

                Journal
                J Mol Biol
                Journal of Molecular Biology
                Elsevier
                0022-2836
                1089-8638
                30 November 2007
                30 November 2007
                : 374
                : 3
                : 749-763
                Affiliations
                [1 ]Department of Veterinary Medicine, Madingley Road, University of Cambridge, Cambridge CB3 0ES, UK
                [2 ]Department of Biochemistry, 80 Tennis Court Road, University of Cambridge, Cambridge CB2 1GA, UK
                [3 ]Department of Molecular and Cellular Biology, University of Guelph, Ontario, Canada N1G 2W1
                [4 ]School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, UK
                Author notes
                [* ]Corresponding author. jking01@ 123456uoguelph.ca
                [†]

                J.D.K. and N.J.H. contributed equally to this work.

                [2]

                Present addresses: J. D. King, Department of Molecular and Cellular Biology, University of Guelph, Ontario, Canada N1G 2W1; N. J. Harmer, School of Biosciences, The Henry Wellcome Building for Biocatalysis, University of Exeter, Stocker Road, Exeter EX4 4QD, UK; A. Preston, Department of Clinical Veterinary Science, University of Bristol, Langford House, Langford BS40 5DU, UK; M. Rejzek and R. A. Field, Department of Biological Chemistry, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK.

                Article
                YJMBI59794
                10.1016/j.jmb.2007.09.055
                2279256
                17950751
                f7685c45-b473-4234-8d6f-9d754805b77f
                © 2007 Elsevier Ltd.

                This document may be redistributed and reused, subject to certain conditions.

                History
                : 4 July 2007
                : 19 September 2007
                : 20 September 2007
                Categories
                Article

                Molecular biology
                sdr, short-chain dehydrogenase/reductase,ump, uridine monophosphate,o-antigen biosynthesis,l-galnac3naca, 2,3-diacetamido-2,3-dideoxy-l-galacturonic acid,mcs, multiple cloning site,dtdp, deoxythymidine diphosphate,udp-d-mannac3naca, udp-2,3-diacetamido-2,3-dideoxy-d-mannuronic acid,blast, basic local alignment search tool,pdb, protein data bank,bordetella,short-chain dehydrogenase/reductase,lps, lipopolysaccharide,lipopolysaccharide,udp-l-galnac3naca, udp-2,3-diacetamido-2,3-dideoxy-l-galacturonic acid,gmer, gdp-4-keto-6-deoxymannose 3,5-epimerase/reductase,gme, gdp-mannose 3,5-epimerase,x-ray crystallography

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