Aromatic amino acid aminotransferase (ArATPh), which has a melting temperature of 120°C, is one of the most thermostable aminotransferases yet to be discovered. The crystal structure of this aminotransferase from the hyperthermophilic archaeon Pyrococcus horikoshii was determined to a resolution of 2.1 Å. ArATPh has a homodimer structure in which each subunit is composed of two domains, in a manner similar to other well characterized aminotransferases. By the least square fit after superposing on a mesophilic ArAT, the ArATPh molecule exhibits a large deviation of the main chain coordinates, three shortened ␣-helices, an elongated loop connecting two domains, and a long loop transformed from an ␣-helix, which are all factors that are likely to contribute to its hyperthermostability. The pyridine ring of the cofactor pyridoxal 5-phosphate covalently binding to Lys 233 is stacked parallel to F121 on one side and interacts with the geminal dimethyl-CH/ groups of Val 201 on the other side. This tight stacking against the pyridine ring probably contributes to the hyperthermostability of ArATPh. Compared with other ArATs, ArATPh has a novel substrate specificity, the order of preference being Tyr > Phe > Glu > Trp > His > > Met > Leu > Asp > Asn. Its relatively weak activity against Asp is due to lack of an arginine residue corresponding to Arg 292 * (where the asterisk indicates that this is a residues supplied by the other subunit of the dimer) in pig cytosolic aspartate aminotransferase. The enzyme recognizes the aromatic substrate by hydrophobic interaction with aromatic rings (Phe 121 and Tyr 59 *) and probably recognizes acidic substrates by a hydrophilic interaction involving a hydrogen bond network with Thr 264 *.

A L L-glycosidase gene homolog of Pyrococcus horikoshii (BGPh) was successfully expressed in Escherichia coli. The enzyme was localized in a membrane fraction and solubilized with 2.5% Triton X-100 at 85³C for 15 min. The optimum pH was 6.0 and the optimum temperature was over 100³C, respectively. BGPh stability was dependent on the presence of Triton X-100, the enzyme's half-life at 90³C (pH 6.0) was 15 h. BGPh has a novel substrate specificity with k cat /K m values high enough for hydrolysis of L L-D-Glcp derivatives with long alkyl chain at the reducing end and low enough for the hydrolysis of L Llinked glucose dimer more hydrophilic than aryl-or alkyl-L L-DGlcp.z 2000 Federation of European Biochemical Societies.

The organization of the phosphate-specific transport (pst) operon in Pseudomonas aeruginosa has been determined. The gene order of the pst operon is pstC, pstA, pstB, phoU, and a well-conserved Pho box sequence (16/18 bases identical) exists in the promoter region. The most striking difference from the known Escherichia coli pst operon is the lack of the pstS gene encoding a periplasmic phosphate (Pi)-binding protein. Even though the three pst genes were absolutely required for P(i)-specific transport, expression of the pst operon at high levels did not increase P(i) uptake in P. aeruginosa. DNA sequences for the pstB and phoU genes have been determined previously. The newly identified pstC and pstA genes encode possible integral membrane proteins of 677 amino acids (M(r) 73,844) and 513 amino acids (M(r) 56,394) respectively. The amino acid sequences of PstC and PstA predict that these proteins contain a long hydrophilic domain not seen in their E. coli counterparts. A chromosomal deletion of the entire pst operon rendered P. aeruginosa unable to repress P(i) taxis under conditions of P(i) excess. The phoU and pstB genes are essential for repressing P(i) taxis. However, mutants lacking either PstC or PstA alone were able to repress P(i) taxis under conditions of P(i) excess.

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