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This Article Appeared in a Journal Published by Elsevier. the Attached This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy http://dx.doi.org/10.1016/j.jmb.2012.08.013 J. Mol. Biol. (2012) 423, 831–846 Contents lists available at www.sciencedirect.com Journal of Molecular Biology journal homepage: http://ees.elsevier.com.jmb Conservation of Functionally Important Global Motions in an Enzyme Superfamily across Varying Quaternary Structures Emily K. Luebbering 1, Jacob Mick 1, Ranjan K. Singh 2, John J. Tanner 1, 2, Ritcha Mehra-Chaudhary 3 and Lesa J. Beamer 1, 2⁎ 1Department of Biochemistry, University of Missouri, Columbia, MO 65211, USA 2Department of Chemistry, University of Missouri, Columbia, MO 65211, USA 3Structural Biology Core, University of Missouri, Columbia, MO 65211, USA Received 3 May 2012; The α‐D‐phosphohexomutase superfamily comprises enzymes involved in received in revised form carbohydrate metabolism that are found in all kingdoms of life. Recent 16 August 2012; biophysical studies have shown for the first time that several of these accepted 17 August 2012 enzymes exist as dimers in solution, prompting an examination of the Available online oligomeric state of all proteins of known structure in the superfamily (11 27 August 2012 different proteins; 31 crystal structures) via computational and experimen- tal analyses. We find that these proteins range in quaternary structure from Edited by A. Panchenko monomers to tetramers, with 6 of the 11 known structures being likely oligomers. The oligomeric state of these proteins not only is associated in Keywords: some cases with enzyme subgroup (i.e., substrate specificity) but also phosphohexomutase; appears to depend on domain of life, with the two archaeal proteins existing normal mode analysis; as higher‐order oligomers. Within the oligomers, three distinct interfaces oligomeric interface; are observed, one of which is found in both archaeal and bacterial proteins. global fluctuations; Normal mode analysis shows that the topological arrangement of the conformational flexibility oligomers permits domain 4 of each protomer to move independently as required for catalysis. Our analysis suggests that the advantages associated with protein flexibility in this enzyme family are of sufficient importance to be maintained during the evolution of multiple independent oligomers. This study is one of the first showing that global motions may be conserved not only within protein families but also across members of a superfamily with varying oligomeric structures. © 2012 Elsevier Ltd. All rights reserved. *Corresponding author. Department of Biochemistry, University of Missouri, Columbia, MO 65211, USA. E-mail address: [email protected]. Abbreviations used: BaPNGM, Bacillus anthracis PNGM; CaPAGM, Candida albicans PAGM; CSS, complexation significance score; DLS, dynamic light scattering; FtPNGM, Francisella tularensis PNGM; NMA, normal mode analysis; OcPGM, Oryctolagus cuniculus PGM; PAGM, N-acetylglucosamine phosphate mutase; PaPMM, Pseudomonas aeruginosa phosphomannomutase/phosphoglucomutase; PDB, Protein Data Bank; PGM, phosphoglucomutase; PMM/PGM, phosphomannomutase/phosphoglucomutase; PNGM, phosphoglucosamine mutase; PhPMM, Pyrococcus horikoshii phosphomannomutase/phosphoglucomutase; SAXS, small‐angle X-ray scattering; St-mutase, Sulfolobus tokodaii phosphohexomutase; StPGM, Salmonella typhimurium PGM; TtPGM, Thermus thermophilus PGM. 0022-2836/$ - see front matter © 2012 Elsevier Ltd. All rights reserved. Author's personal copy 832 Conservation of Global Motions in Enzymes Introduction Herein, we analyze the oligomeric state of all known structures of enzymes from this superfamily The α‐D‐phosphohexomutase enzyme superfam- currently found in the Protein Data Bank (PDB), ily is ubiquitous in organisms from all kingdoms of representing a total of 11 proteins and 31 crystal life. Enzymes in this superfamily catalyze the structures. Crystal packing analyses and biophysical reversible conversion of phosphosugar substrates, characterization show that more than half of these from the 1-phospho to 6-phospho form. Four proteins are likely oligomers, with the two archaeal subgroups of the superfamily have been well enzymes adopting the largest tetrameric assembly. characterized: phosphoglucomutase (PGM), phos- Two distinct dimeric arrangements that are specific phomannomutase/phosphoglucomutase (PMM/ to different subgroups of the superfamily are PGM), phosphoglucosamine mutase (PNGM), and observed, suggesting independent evolutionary N-acetylglucosamine phosphate mutase (PAGM).1,2 origins. The fluctuation dynamics of the various Although all proteins in the superfamily catalyze oligomers were characterized using normal mode the same reaction, they have differing preferences analysis (NMA). For all assemblies, the most mobile for the sugar moiety of the substrate, as implied by region of the protein is domain 4, consistent with the names of the various subgroups. Due to their different conformers of this domain observed in roles in numerous biosynthetic and metabolic various crystal structures. The conservation of low‐ pathways, including those involved in virulence frequency global motions characterized in this study of human pathogens, many of these enzymes are of is consistent with the known mechanistic impor- 3–6 interest as potential drug targets and may also tance of conformational change of the α‐D‐phospho- – have utility in metabolic engineering.7 10 hexomutases. This study supports recent evidence The reaction mechanism of the α‐D‐phosphohex- for the evolutionary conservation of protein vibra- omutases involves two successive phosphoryl tional dynamics in homologous proteins27 but – transfers.11 15 Initially, the enzyme donates a extends this concept to include a superfamily with phosphoryl group from a conserved active‐site varying quaternary structures. Thus, evolutionary phosphoserine residue to substrate, forming a pressure to maintain conformational flexibility bisphosphorylated sugar intermediate. The inter- across varying molecular shapes may be a factor mediate then reorients in the active site and must affecting the evolution of oligomers in other protein rebind in the opposite orientation so that the serine families. can accept the alternate phosphoryl group from the intermediate, forming product and regenerating active, phosphorylated enzyme. Structural studies Results have shown that conformational change of the enzyme is required at several points in the multi- step reaction, including upon binding of substrate, Overview of the superfamily and structure of the to permit reorientation of the intermediate, and for protomers – the release of product.16 19 Over the last two decades, crystal structures Enzymes in the α‐D‐phosphohexomutase super- have been determined for at least one protein in family are ubiquitous in all organisms including 1 each subgroup of the α‐D‐phosphohexomutase bacteria, archaea, and eukaryotes. They participate – superfamily.20 24 These studies have shown that in a variety of key biosynthetic and metabolic theenzymesshareaconservedfour-domain pathways, determined by the specificity for the architecture, with a large, centrally located active‐ sugar moiety of their substrate. The PGM proteins site cleft. Another feature commonly observed in have high specificity for glucose; the PMM/PGMs the crystal structures is conformational variability can utilize either glucose or mannose; the PNGMs of the C-terminus, which moves via a hinge-type prefer glucosamine; and the PAGMs utilize N- rotation relative to the rest of the protein, and is acetylglucosamine phosphate. Additional specific- correlated with ligand binding.16,18,20,21,25,26 Until ities/activities have been reported for some proteins recently, the quaternary structures of these pro- in the superfamily, including phosphopentomutase teins had been largely unexamined, perhaps and glucose 1,6-bisphosphate synthase activity.28 because the best characterized enzymes were The currently available crystal structures for known to be monomers. However, biochemical enzymes in the α‐D‐phosphohexomutase super- characterization of Salmonella typhimurium PGM family include 11 proteins from 10 different (StPGM) and Bacillus anthracis PNGM (BaPNGM) organisms, and a total of 31 crystal structures demonstrated that these two proteins exist as (Table 1). These structures reflect the widespread dimers in solution.20,21,25 The discovery of oligo- phylogenetic distribution of these proteins, with mers within α‐D‐phosphohexomutase superfamily six from bacteria, two from archaea, and three prompted the current examination of all known from eukaryotes. Sequence comparisons of these structures of these enzymes. proteins show profound diversity, consistent with Author's personal copy Conservation of Global Motions in Enzymes 833 Table 1. Summary of α‐D‐phosphohexomutase of known structure Subgroup PISA (EC no.) Organism
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