No abstract

The protein TA0175 has a large number of sequence homologues, most of which are annotated as unknown and a few as belonging to the haloacid dehalogenase superfamily, but has no known biological function. Using a combination of amino acid sequence analysis, three-dimensional crystal structure information, and kinetic analysis, we have characterized TA0175 as phosphoglycolate phosphatase from Thermoplasma acidophilum. The crystal structure of TA0175 revealed two distinct domains, a larger core domain and a smaller cap domain. The large domain is composed of a centrally located five-stranded parallel ␤-sheet with strand order S10, S9, S8, S1, S2 and a small ␤-hairpin, strands S3 and S4. This central sheet is flanked by a set of three ␣-helices on one side and two helices on the other. The smaller domain is composed of an open faced ␤-sandwich represented by three antiparallel ␤-strands, S5, S6, and S7, flanked by two oppositely oriented ␣-helices, H3 and H4. The topology of the large domain is conserved; however, structural variation is observed in the smaller domain among the different functional classes of the haloacid dehalogenase superfamily. Enzymatic assays on TA0175 revealed that this enzyme catalyzed the dephosphorylation of phosphoglycolate in vitro with similar kinetic properties seen for eukaryotic phosphoglycolate phosphatase. Activation by divalent cations, especially Mg 2؉ , and competitive inhibition behavior with Cl ؊ ions are similar between TA0175 and phosphoglycolate phosphatase. The experimental evidence presented for TA0175 is indicative of phosphoglycolate phosphatase.

ATP:cobalamin adenosyltransferase MMAB was recently identified as the gene responsible for a disorder of cobalamin metabolism in humans (cblB complementation group). The crystal structure of the MMAB sequence homologue from Thermoplasma acidophilum (TA1434; GenBank TM identification number giͦ16082403) was determined to a resolution of 1.5 Å. TA1434 was confirmed to be an ATP:cobalamin adenosyltransferase, which depended absolutely on divalent metal ions (Mg 2؉ > Mn 2؉ > Co 2؉ ) and only used ATP or dATP as adenosyl donors. The apparent K m of TA1434 was 110 M (k cat ‫؍‬ 0.23 s ؊1 ) for ATP, 140 M (k cat ‫؍‬ 0.11 s ؊1 ) for dATP, and 3 M (k cat ‫؍‬ 0.18 s ؊1 ) for cobalamin. TA1434 is a trimer in solution and in the crystal structure, with each subunit composed of a five-helix bundle. The location of diseaserelated point mutations and other residues conserved among the homologues of TA1434 suggest that the active site lies at the junctions between the subunits. Mutations in TA1434 that correspond to the disease-related mutations resulted in proteins that were inactive for ATP:cobalamin adenosyltransferase activity in vitro, confirming that these mutations define the molecular basis of the human disease. Cobalamin (vitamin B 12 ) is an important cofactor in both prokaryotes and eukaryotes. A wide variety of cobalamin-dependent metabolic reactions are known, including acetyl-CoA synthesis, generation of deoxyribonucleotides for DNA synthesis, methane production in methanogenic archaea, and fermentation of small molecules (1). The metabolic requirement for cobalamin varies among different organisms. Some bacteria, such as Escherichia coli, use the vitamin but do not synthesize it (2), whereas other bacteria have no need for it (1). Animals require cobalamin but must rely on dietary sources, and it is believed that plants and fungi neither synthesize nor use cobalamin (1, 3). A characteristic that distinguishes cobalamin from all other vitamins is that only certain bacteria and archaea possess the ability for its de novo synthesis (3, 4).Prior to its use in the cell, cobalamin must be metabolized into one of two biologically active forms, adenosylcobalamin (AdoCbl) 1 and methylcobalamin (MeCbl). In humans, these cofactors are known to be used in two enzyme systems. The first enzyme, methionine synthase, uses MeCbl to produce methionine from homocysteine (2, 5). The second enzyme, methylmalonyl-CoA mutase, uses AdoCbl as a coenzyme to convert methylmalonyl-CoA to succinyl-CoA, which can then enter the trichloroacetic acid cycle (6, 7). This AdoCbl-dependent reaction is important in the catabolism of methionine, valine, threonine, isoleucine, odd chain fatty acids, and cholesterol (8, 9).Cobalamin itself comprises a central corrin ring that provides four ligands for a hexacoordinated cobalt ion (3, 4). The conversion of cobalamin to AdoCbl and MeCbl involves the reduction of the cobalt ion from a Co(III) oxidation state to Co(I) and then the addition of the functional ligand (2, 10). In the formation of AdoCbl, the 5...

No abstract