Glycinamide ribonucleotide synthetase (GAR-syn, PurD) catalyses the second reaction of the purine biosynthetic pathway; the conversion of phosphoribosylamine, glycine and ATP to glycinamide ribonucleotide (GAR), ADP and Pi. In the present study, crystal structures of GAR-syn's from Thermus thermophilus, Geobacillus kaustophilus and Aquifex aeolicus were determined in apo forms. Crystal structures in ligand-bound forms were also determined for G. kaustophilus and A. aeolicus proteins. In general, overall structures of GAR-syn's are similar to each other. However, the orientations of the B domains are varied among GAR-syn's and the MD simulation suggested the mobility of the B domain. Furthermore, it was demonstrated that the B loop in the B domain fixes the position of the β- and γ- phosphate groups of the bound ATP. The structures of GAR-syn's and the bound ligands were compared with each other in detail, and structures of GAR-syn's with full ligands, as well as the possible reaction mechanism, were proposed.

An alcohol dehydrogenase (ADH) gene, ST0053, from Sulfolobus tokodaii was expressed in Escherichia coli. The purified recombinant enzyme was an NAD-dependent medium-chain ADH with high thermostability and tolerance of a wide range of pHs. This is the first step in creating an experimental functionality library of 10 genes annotated as ADHs in the S. tokodaii genome.NAD(P)-dependent alcohol dehydrogenases (ADHs; EC 1.1.1.1 and EC 1.1.1.2) catalyze the reversible oxidation of alcohols to their corresponding aldehydes or ketones. Many kinds of ADHs have been identified in a variety of microorganisms and are utilized for the production and detection of alcohols, aldehydes, and ketones (1, 11). Particularly useful are ADHs from thermophiles and hyperthermophiles, which are often more stable than their counterparts from mesophiles and psychrophiles (6, 7). These thermostable enzymes provide us with long-term functionality and novel reaction systems, such as the enzymatic vapor reaction under high temperature. Ten different genes have been annotated as NAD(P)-dependent ADHs in the genome of the hyperthermophilic and acidophilic archaeon Sulfolobus tokodaii strain 7 (13). We have been interested in compiling the structural and functional characteristics of their products into a library. Such a library would provide much useful information that could facilitate their application. We here describe the expression of ST0053, one of the 10 ADH genes in the S. tokodaii genome, and the subsequent purification and characteristics of its product.Cloning and expression of ST0053. A hybrid DNA plasmid, pET11a/ST0053, whose ST0053 open reading frame was amplified by PCR and inserted into the NdeI-BamHI site of pET11a (Novagen), was provided by Seiki Kuramitsu (Department of Biology, Graduate School of Science, Osaka University, Osaka, Japan). The plasmid was transformed into E. coli strain Rosetta(DE3), after which the transformants were cultured for 16 h in 10 ml of Luria-Bertani medium containing ampicillin (50 g ml Ϫ1 ) at 37°C with shaking at 120 rpm. This culture medium was then added to 1 liter of Luria-Bertani medium containing the same amount of antibiotic in a 2-liter Erlenmeyer flask and incubated at 37°C with shaking at 220 rpm. Once the optical density at 660 nm reached 0.5, expression was induced by the addition of isopropyl-␤-D-thiogalactopyranoside (IPTG; 1 mM final concentration) and cultivation was continued at 37°C for an additional 4 h. The culture was harvested by centrifugation at 10,000 ϫ g for 10 min at 4°C, the cells were washed with 0.85% NaCl and pelleted, and then the cell pellet was stored at Ϫ80°C.Purification. To purify ADH, the stored cells (wet weight, about 2.55 g) were suspended in 10.2 ml of 20 mM Tris-HCl buffer (pH 8.0) containing 0.01% 2-mercaptoethanol (2-ME) (buffer A) and then disrupted by ultrasonication on ice (Tomy Ultrasonic Disruptor UD-201 set at an output level of 2 and a 50% duty cycle; three sonications for 3 min each with 5-min intervals). The resultant homogenate was centrifuged a...