Abstract
GMP catalyzes the formation of GDP-Man, a fundamental precursor for protein glycosylation and bacterial cell wall and capsular polysaccharide biosynthesis. Crystal structures of GMP from the thermophilic bacterium Thermotoga maritima in the apo form, in complex with the substrates mannose-1-phosphate or GTP and bound with the end product GDP-Man in the presence of the essential divalent cation Mg(2+), were solved in the 2.1-2.8 A resolution range. The T. maritima GMP molecule is organized in two separate domains: a N-terminal Rossman fold-like domain and a C-terminal left-handed beta-helix domain. Two molecules associate into a dimer through a tail-to-tail arrangement of the C-terminal domains. Comparative analysis of the structures along with characterization of enzymatic parameters reveals the bases of substrate specificity of this class of sugar nucleotidyltransferases. In particular, substrate and product binding are associated with significant changes in the conformation of loop regions lining the active center and in the relative orientation of the two domains. Involvement of both the N- and C-terminal domains, coupled to the catalytic role of a bivalent metal ion, highlights the catalytic features of bacterial GMPs compared with other members of the pyrophosphorylase superfamily.
Highlights
Nucleoside-5Ј-diphosphosugars (NDP-sugars),3 referred to as sugar nucleotides, represent the most common form of activated donor substrates used by glycosyltransferases in various biosynthetic pathways
A phosphomannose isomerase (PMI) first converts fructose-6phosphate to mannose-6-phosphate, which is converted to mannose-1-phosphate (Man1P) by a phosphomannomutase
The sequences from mono- and bifunctional PMIs, classified as PMI of type I and II, respectively, lack significant overall homology, the sequence associated to GMP activity has been conserved during the course of evolution [4]
Summary
Expression, and Purification—TmGMP, TM1033 (GDP-mannose pyrophosphorylase/mannose-1-phosphate guanylyltransferase; UniProt Q9X0C3) was amplified by PCR from T. maritima, strain MSB8, genomic DNA using Pfu Turbo (Stratagene) and primer pairs encoding the predicted 5Ј- and 3Ј-ends of TmGMP. Larger crystals were grown in hanging drops by mixing equal volumes of protein (20 mg/ml in 10 mM Tris, pH 8.0, 150 mM NaCl) and reservoir (35% (v/v) MPD, 0.1 M phosphate citrate, pH 7.5) solutions. Crystallization of the TmGMP-Man1P complex was achieved in sitting drops using a protein to well solution ratio of 3:1 and 35% MPD as the well solution. For the TmGMP-GDP-Man complex, sitting drops were set up by mixing equal volumes of the protein solution and well solution made of 30% MPD, 0.1 M sodium acetate, pH 4.6, 20 mM MgCl2. Crystals of the TmGMP GTP complex were obtained in hanging drops with a protein to well solution ratio of 2:1 and 30% MPD, 0.1 M sodium acetate, pH 5.0, 20 mM MgCl2 as the well solution. The data were collected on ESRF (Grenoble, France) and SOLEIL (Saint-Aubin, France) beamlines, processed with MOSFLM [13] or XDS [14], and scaled and merged with SCALA [15]
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