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Instruments / Technical Area	Publication Details	Published	Actions
I04-1-Macromolecular Crystallography (fixed wavelength)	Structural and functional characterization of NanU, a novel high-affinity sialic acid-inducible binding protein of oral and gut-dwelling Bacteroidetes species Chatchawal Phansopa , Sumita Roy , J B Rafferty , C. W. ian Douglas , Jagroop Pandhal , Phillip C. Wright , David J Kelly , Graham p. Stafford Biochemical Journal 458, 499 - 511 DOI: 10.1042/BJ20131415 PMID: 24351045 Diamond Proposal Number(s): [8987, 7423] Open Access Show Abstract ▼ Abstract: Many human-dwelling bacteria acquire sialic acid for growth or surface display. We identified previously a sialic acid utilization operon in Tannerella forsythia that includes a novel outer membrane sialic acid-transport system (NanOU), WHERE NanO (neuraminate outer membrane permease) is a putative TonB-dependent receptor and NanU (extracellular neuraminate uptake protein) is a predicted SusD family protein. Using heterologous complementation of nanOU genes into an Escherichia coli strain devoid of outer membrane sialic acid permeases, we show that the nanOU system from the gut bacterium Bacteroides fragilis is functional and demonstrate its dependence on TonB for function. We also show that nanU is required for maximal function of the transport system and that it is expressed in a sialic acid-responsive manner. We also show its cellular localization to the outer membrane using fractionation and immunofluorescence experiments. Ligand-binding studies revealed high-affinity binding of sialic acid to NanU (Kd ~400 nM) from two Bacteroidetes species as well as binding of a range of sialic acid analogues. Determination of the crystal structure of NanU revealed a monomeric SusD-like structure containing a novel motif characterized by an extended kinked helix that might determine sugar-binding specificity. The results of the present study characterize the first bacterial extracellular sialic acid-binding protein and define a sialic acid-specific PUL (polysaccharide utilization locus).	Mar 2014
	Structural explanation for the role of Mn2+ in the activity of phi 6 RNA-dependent RNA polymerase Minna M. Poranen , Paula Salgado , Minni R. L. Koivunen , Sam Wright , Dennis H. Bamford , David I. Stuart , Jonathan M. Grimes Nucleic Acids Research 36 (20), 6633-6644 DOI: 10.1093/nar/gkn632 Show Abstract ▼ Abstract: The biological role of manganese (Mn2+) has been a long-standing puzzle, since at low concentrations it activates several polymerases whilst at higher concentrations it inhibits. Viral RNA polymerases possess a common architecture, reminiscent of a closed right hand. The RNA-dependent RNA polymerase (RdRp) of bacteriophage {phi}6 is one of the best understood examples of this important class of polymerases. We have probed the role of Mn2+ by biochemical, biophysical and structural analyses of the wild-type enzyme and of a mutant form with an altered Mn2+-binding site (E491 to Q). The E491Q mutant has much reduced affinity for Mn2+, reduced RNA binding and a compromised elongation rate. Loss of Mn2+ binding structurally stabilizes the enzyme. These data and a re-examination of the structures of other viral RNA polymerases clarify the role of manganese in the activation of polymerization: Mn2+ coordination of a catalytic aspartate is necessary to allow the active site to properly engage with the triphosphates of the incoming NTPs. The structural flexibility caused by Mn2+ is also important for the enzyme dynamics, explaining the requirement for manganese throughout RNA polymerization.	Oct 2008

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