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 sh own to encode one of the pristinamycin I (PI) synthetases. Analysis of the homologies observed from the deduced amino acid sequences suggest ed 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 cultu re medium. Further confirmation was obtained when papM was overexpress ed in Escherichia coli and the corresponding protein purified to homog eneity. 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.