Crystal structure of uronate isomerase (TM0064) from <i>Thermotoga maritima</i> at 2.85 Å resolution

Abstract

The TM0064 gene of Thermotoga maritima encodes a predicted uronate isomerase (EC 5.3.1.12) with a molecular weight of 52,174 Da and a calculated isoelectric point of 5.68. Uronate dehydrogenase catalyzes the conversion of D-glucuronate to D-fructuronate, or D-galacturonate to D-tagaturonate, which are the first steps in the pathway of glucuronic and galacturonic acid metabolism. This enzyme has no other paralogs, but a number of orthologs have been identified in other bacterial species. Here, we report the crystal structure of TM0064 determined with use of the semiautomated high-throughput pipeline of the Joint Center for Structural Genomics.1 This is the first structure of a protein from this family to be determined. We solved the structure of TM0064 to 2.85 Å resolution using the multiple-wavelength anomalous dispersion (MAD) method. Data collection, model, and refinement statistics are summarized in Table I.The final model includes residues 1–450 for each of the three independent molecules in the asymmetric unit (the C-terminal Gly is not present in the final model), and 107 water molecules. The Matthews coefficient (Vm) for TM0064 is 2.73 Å3/Da, and the estimated solvent content is 58.9%. The Ramachandran plot produced by PROCHECK 3.42 shows that 90% of the residues are in the most favored regions, 9.8% in additional allowed regions, and 0.2% in generously allowed regions. No residues lie in disallowed regions. The TM0064 monomer consists of a single polypeptide chain of 451 amino acids composed of 25 helices (21 α-helices, and four 310-helices), and 9 β-strands [Fig. 1(A, C, and E)]. (A) Domain organization of Thermotoga maritima TM0064 uronate isomerase. Domain A contains two noncontiguous segments (yellow and blue). (B) Solvent accessible surface (1.4 Å probe radius) of TM0064. The red arrow indicates the channel leading to the active site. The black arrow indicates the position of the putative metal ion. (C) Ribbon representation showing the distribution of secondary structure elements in TM0064. Strands are shown in cyan and helices, in red. The arrow points at the active site. (D) Close-up view of the active site, showing residues interacting with the putative metal ion. (E) Diagram showing the secondary structure elements in TM0064 superimposed to its primary sequence. Residues interacting with the putative metal ion are indicated by green triangles. Helices are labeled H1–H25. Strands are labeled according to their respective β-sheets (i.e., A and B). The location of the β-hairpin formed by β-strands 6 and 7 (labeled C), as well as the location of β and γ turns, are also depicted in the diagram. The total α-helix, 310-helix, and β-strand content is 58.8, 2.4, and 7.8%, respectively [Fig. 1(C)]. The 9 β-strands form two parallel β-sheets [strands labeled A and B in Fig. 1(E)] and a β-hairpin (β6–β7) [strands labeled C in Fig. 1(E)]. The first β-sheet is formed by strands β1–β3, with a topology 1X 1X. The second β-sheet (B) is formed by strands β4, β5, β8, and β9, with a 1X 1X 1X topology. The predicted active site is defined by residues His30, His32, Trp366, and Asp397, which coordinate a putative metal ion that has been conservatively modeled as water 1 in the absence of any experimental data on the nature of the metal [Fig. 1(D)]. This finding is consistent with observations indicating that uronate isomerases are capable of binding metals, such as Zn2+ or Cu2+, which can act as inhibitors.3 The activity of this enzyme has been characterized in three different organisms: Escherichia coli,3 Erwinia carotovora,4 and Flavobacterium heparinum.5 Twenty homologs belonging to this protein family have been identified in Bacteria, including Thermotogales, Cyanobacteria, Proteobacteria (Alpha and Gamma subdivisions), and Firmicutes (all in the Bacillus/Clostridium group). This enzyme family is absent in Archaea and Eukaryotes. Homology structural models of all these bacterial homologs can be accessed at http://www1.jcsg.org/