The ribose binding protein of Salmonella typhimurium has been shown to be the receptor for ribose

Rat liver cDNA libraries constructed in Xgt11 were screened for reactivity with polyclonal antibodies to native S-adenosyl-L-homocysteine (AdoHcy) hydrolase (adenosylhomocysteinase; EC 3.3.1.1). Five clones were isolated and sequenced. The amino acid sequence, deduced from the cDNA sequence, contained the sequence of eight peptides obtained by tryptic and cyanogen bromide fragmentation of rat liver AdoHcy hydrolase. Identification of the amino-and carboxylterminal peptides in the amino acid sequence showed that the complete sequence was obtained. A "fingerprint" sequence was found that is characteristic of dinucleotide-binding domains of many proteins. For AdoHcy hydrolase, this region from the lysine at position 213 to the aspartate at position 244, containing the sequence Gly-Xaa-Gly-Xaa-Xaa-Gly at positions 219-224, is presumably the site of binding for NAD', which is required for the activity of the enzyme.

The genetic locus ahcY, encoding the enzyme S-adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) from the bacterium Rhodobacter capsulates, has been mapped by mutational analysis to within a cluster of genes involved in regulating the induction and maintenance of the bacterial photosynthetic apparatus. Sequence analysis demonstrates that ahcY encodes a 51-kDa polypeptide that displays 64% sequence identity to its human homolog. Insertion mutants in ahcY lack detectable S-adenosyl-L-homocysteine hydrolase activity and, as a consequence, S-adenosyl-L-homocysteine accumulates in the cells, resulting in a 16-fold decrease in the intracellular ratio of S-adenosyl-L-methionine to S-adenosyl-L-homocysteine as compared to wild-type cells. The ahcY disrupted strain fails to grow in minimal medinm; however, growth is restored in minimal medium supplemented with methionine or homocysteine or in a complex medium, thereby indicating that the hydrolysis of S-adenosyl-L-homocysteine plays a key role in the metabolism of sulfur-containing amino acids. The ahcY mutant, when grown in supplemented medium, synthesizes significantly reduced levels of bacteriochlorophyll, indicating that modulation of the intracellular ratio of S-adenosyl-L-methionine to S-adenosyl-L-homocysteine may be an important factor in regulating bacteriochlorophyll biosynthesis.S-Adenosyl-L-homocysteine hydrolase (AdoHcyase, EC 3.3.1.1) was first described in rat liver extracts as the activity responsible for the reversible hydrolysis of S-adenosyl-Lhomocysteine (AdoHcy) to adenosine and homocysteine ( Fig. 1) (1). AdoHcy is formed as a direct product of transmethylation reactions involving S-adenosyl-L-methionine (AdoMet) (2) and is known to be a potent inhibitor of most AdoMet-mediated methyl-transfer reactions.AdoHcyase has been found in all cells that have been tested, with the exception of Escherichia coli and certain related bacteria (3-5). In the latter, the hydrolysis ofAdoHcy to adenine and homocysteine requires two enzymatic steps catalyzed by a specific AdoHcy nucleosidase (6) and S-ribosyl-L-homocysteine hydrolase (7). AdoHcyase isolated from various sources always consists of a number of identical subunits, each containing 1 mol of tightly bound NAD+ (8).The amino acid sequences of the rat liver, human placenta, and Dictyostelium enzymes have been deduced from cDNA sequences and show a high degree of sequence conservation (9-11). Site-directed mutational analyses coupled with in vitro biochemistry have demonstrated the presence of a nucleotide-binding domain (12, 13), but further conclusions regarding the protein structure have been hindered by lack of significant sequence diversity and a convenient system for genetic analysis.

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When WBC264-9C cells are preincubated with pertussis toxin, chemotaxis is inhibited and ADPribosylation of a membrane protein with a subunit Mr 41,000 is observed. Both the inhibition of chemotaxis and the ADPribosylation by pertussis toxin display a similar time lag, temperature dependence, and pertussis toxin-concentration dependence. Although the inhibition of chemotaxis and the ADP-ribosylation of the membrane protein are qualitatively correlated, nearly complete inhibition of chemotaxis occurs when there is only partial ADP-ribosylation of the membrane protein. Pertussis toxin-catalyzed ADP-ribosylation of the Mr 41,000 protein in WBC264-9C membranes is stimulated by GDP, GTP, and to a lesser extent by GMP; the nonhydrolyzable GTP analog guanosine 5'-[fB,-imidoltriphosphate has no effect. WBC264-9C membranes have a high-affinity GTPase activity, which is partially inhibited in membranes from pertussis toxin-treated cells. Neither GTPase activity nor adenylate cyclase activity in membranes from WBC264-9C cells is affected by fMet-Leu-Phe, an attractant for these cells. Our results suggest that a guanine nucleotide binding protein may be involved in chemotaxis, but they do not indicate an involvement of adenylate cyclase.