Etude de deux enzymes bacteriennes : mecanisme de la glucosamine-6-P synthase d'Escherichia coli au niveau du site fructose-6-P, purification d'une chorismate mutase monofonctionnelle chez Bacillus subtilis

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Possible involvement of Lys603 from Escherichia coli glucosamine-6-phosphate synthase in the binding of its substrate fructose 6-phosphate

International audiencePyridoxal 5'-phosphate is a competitive inhibitor of glucosamine-6-phosphate synthase with respect to the substrate fructose 6-phosphate. Irreversible inactivation of pyridoxal-5'-phosphate-treated enzyme with ['"CJcyanide resulted in covalent incorporation of close to 1 mol pyridoxal 5'-phosphate/mol enzyme subunit. The enzyme-pyridoxaL5'-phosphate complex could also be inactivated by reduction with NaBH,CN. Sequence analysis of the unique radioactively labelled tryptic peptide, resulting from inactivation with [,H]NaBH,CN, identified the C-terminal nonapeptide encompassing the modified Lys603. The presence of fructose 6-phosphate protected this residue from pyridoxylation. Direct evidence that a lysine residue is involved in the binding of the substrate as a Schiff base came from the isolation at 4°C of a enzyme-fructose-6-phosphate complex in a 1 :1 molar ratio. Treatment of the enzyme-['4C]fructose-6-phosphate complex with NaBH,CN revealed one site of modification in the tryptic peptide map. In contrast, trapping the same complex with potassium cyanide resulted in the isolation of several radiolabelled peptides containing lysines which could potentially bind fructose 6phosphate. However, since the radioactivity was not specifically associated with the lysine residues, it is suggested that these I4C-labelled peptides resulted from the decomposition of an unstable a,a'-dihydroxyaminonitrile adduct rather than from a lack of specificity of fructose 6-phosphate fixation. Lys603 is then the candidate of choice for fructose 6-phosphate binding since it lies at or near the active site as demonstrated by the trapping experiments with pyridoxal 5'-phosphate described above, and among the lysines which belong to the sugar-binding domain this is the only one conserved between the three members of the purF, glutamine-dependent, amidotransferase subfamily which include the glucosamine-6-phosphate synthase from Escherichia coli, Saccharomyces cerevisiae and the Rhizobium nodulation protein NodM

Monofunctional chorismate mutase from Bacillus subtilis: purification of the protein, molecular cloning of the gene, and overexpression of the gene product in Escherichia coli

The monofunctional chorismate mutase from Bacillus subtilis has been purified 2200-fold to homogeneity. The enzyme is a homodimer of subunit Mr = 14,500 and is the smallest natural chorismate mutase that has been characterized. The purified enzyme follows Michaelis-Menten kinetics with a Km of 100 microM and a kcat of 50 s-1, carries no other associated enzymic activities, and is unaffected by any of the aromatic amino acids. The N-terminal amino acid sequence of the protein has been determined, and this information has been used to construct a precise oligonucleotide probe for the gene by means of in vitro DNA amplification from total chromosomal DNA by the polymerase chain reaction. The cloned aroH gene encodes a protein of 127 amino acid residues and is expressed in Escherichia coli. The cloned gene product is indistinguishable from that purified from Bacillus. The aroH coding region was directly subcloned into a phagemid expression vector by means of the polymerase chain reaction. The resulting construct, with the aroH gene positioned behind efficient transcription and translation initiation sequences of E. coli, results in the production of the monofunctional mutase at levels of 30-35% of the soluble cell protein in E. coli transformants. Chorismate mutases comprise a set of functionally related proteins that show little sequence similarity to each other. This diversity stands in contrast to other chorismate-utilizing enzymes