Nathalie Bamas-Jacques

Identification and analysis of genes from Streptomyces pristinaespiralis encoding enzymes involved in the biosynthesis of the 4‐dimethylamino‐l‐phenylalanine precursor of pristinamycin I

Four pap genes (papA, papB, papC, papM ) were found by sequencing near to snbA, a Streptomyces pristinaespiralis gene which was previously shown to encode one of the pristinamycin I (PI) synthetases. Analysis of the homologies observed from the deduced amino acid sequences suggested that these four genes could be involved in the biosynthesis of the PI precursor 4‐dimethylamino‐l‐phenylalanine (DMPAPA). This was first verified when disruption of papA in S. pristinaespiralis led to a PI− phenotype, which was reversed by the addition of DMPAPA into the culture medium. Further confirmation was obtained when papM was overexpressed in Escherichia coli and the corresponding protein purified to homogeneity. It catalysed the two successive N‐methylation steps of 4‐amino‐l‐phenylalanine leading to DMPAPA via 4‐methylamino‐l‐phenylalanine. These results allowed us to assign a function to each of the four pap genes and to propose a biosynthetic pathway for DMPAPA.

Escherichia coli FolC Structure Reveals an Unexpected Dihydrofolate Binding Site Providing an Attractive Target for Anti-microbial Therapy

In some bacteria, such as Escherichia coli, the addition of l-glutamate to dihydropteroate (dihydrofolate synthetase activity) and the subsequent additions of l-glutamate to tetrahydrofolate (folylpolyglutamate synthetase (FPGS) activity) are catalyzed by the same enzyme, FolC. The crystal structure of E. coli FolC is described in this paper. It showed strong similarities to that of the FPGS enzyme of Lactobacillus casei within the ATP binding site and the catalytic site, as do all other members of the Mur synthethase superfamily. FolC structure revealed an unexpected dihydropteroate binding site very different from the folate site identified previously in the FPGS structure. The relevance of this site is exemplified by the presence of phosphorylated dihydropteroate, a reaction intermediate in the DHFS reaction. L. casei FPGS is considered a relevant model for human FPGS. As such, the presence of a folate binding site in E. coli FolC, which is different from the one seen in FPGS enzymes, provides avenues for the design of specific inhibitors of this enzyme in antimicrobial therapy.