Biosynthesis of puromycin by Streptomyces alboniger: Characterization of puromycin N-acetyltransferase

Abstract

Puromycin N-acetyltransferase from Streptomyces alboniger inactivates puromycin by acetylating the amino position of its tyrosinyl moiety. This enzyme has been partially purified by column chromatography through DEAE-cellulose and Affigel Blue and characterized. It has an Mr of 23 000, as determined by gel filtration. In addition to puromycin, the enzyme N-acetylates O-demethylpuromycin, a toxic precursor of the antibiotic, and chryscandin, a puromycin analogue antibiotic. The Km values for puromycin and O-demethylpuromycin are 1.7 and 4.6 microM, respectively. The O-demethylpuromycin O-methyltransferase from S. alboniger, which apparently catalyzes the last step in the biosynthesis of puromycin [Rao, M. M., Rebello, P. F., & Pogell, B. M. (1969) J. Biol. Chem. 244, 112-118], also O-methylates N-acetyl-O-demethylpuromycin. The Km values of the methylating enzyme for O-demethylpuromycin and N-acetyl-O-demethylpuromycin are 260 and 2.3 microM, respectively. These findings suggest that O-demethylpuromycin, if present in S. alboniger, would be N-acetylated and then O-methylated to be converted into N-acetylpuromycin. It might even be possible that N-acetylation of the puromycin backbone takes place at an earlier precursor.

The pur8 gene from the pur cluster of Streptomyces alboniger encodes a highly hydrophobic polypeptide which confers resistance to puromycin

“…4 and data not shown). Transmembrane proteins involved in transport and/or drug resistance contain multiple (12)(13)(14) transmembrane regions [6,41]. These similarities are extended to four partially conserved motifs which are present in Pur8 at positions 49 and 50, 82-94, 117-129 and 163-172 [6,331.…”

“…Moreover, the K,,, (260 pM) of the O-demethylpuromycin 0-methyltransferase for 0-demethylpuromycin is two orders of magnitude higher than the K , (2.3 pM) for Nacetyl-O-demethylpuromycin. It was proposed, therefore, that an earlier precursor (Nh, N6, 0-tridemethylpuromycin) was acetylated by PAC and that, following this step, the biosynthesis of puromycin proceeded through inactive intermediates [12]. Culture filtrates from either S. alboniger or S. lividans transformants incorporating the pur cluster contain small amounts of N-acetylpuromycin and harbour an N-acetylpuromycin N-acetylhydrolase (NAPH) activity which converts this precursor in puromycin.…”

Fluorescent Labeling of SNAP-Tagged Proteins in Cells

“…Puromycin is an aminonucleoside antibiotic, which causes premature chain termination during translation. Resistance gene encodes puromycin N -acetyltransferase which inactivates cytotoxic puromycin by acetylating it [ 32 ].…”

A single plasmid transfection that offers a significant advantage associated with puromycin selection, fluorescence-assisted cell sorting, and doxycycline-inducible protein expression in mammalian cells

“…Puromycin is an aminonucleoside antibiotic produced by Streptomyces alboniger, which causes premature chain termination during translation in various cell types (De La Luna and Ortin 1992;Vara et al 1985). PURO inactivates cytotoxic puromycin by acetylating the amino position of its tyrosinyl moiety (Vara et al 1985).…”