Gene. 40 (1985) 9-14 Elsevier GENE 1431 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA A common precursor for the two subunits of the penicillin acylase from Escherid& zyxwvutsrqponmlkjihgfedcbaZ edi ATCCll105 (R~ombin~t DNA; protein processing; leader peptide; DNA sequence; pBR339 vector) Guillermo Oliver, Fernando Valle, Francisco Rosetti, Marisa Gbmez-Pedrozo, Patricia Santamaria, Guillermo Gosset and Francisco Bolivar * zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Departatzento de Gene&a y Biologia Molecular, Centro de Invest&a&n sobre Ingenieriu Genkticay Biotecnobgia, Universidad Nacionul Autcinoma de M&k>, Cuernavaca, Morelos [MPxicoj Tel. 15273j 172399 (Received May 17th. 1985) (Revision received June iOth, 1985) (Accepted June l7th, 1985) SUMMARY Penicillin acylase (PA) is an industrial enzyme that is zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPON used to convert penicillin G into a precursor for semisynthetic penicillins. We have cloned a segment of DNA that codes for the two subunits required for PA activity. We also report the nucleotide sequence of a DNA fragment that codes for (i) the small subunit, {ii) the N-terminal region of the large subunit and (iii) a putative connecting peptide. These results confirm the existence of a common precursor for both peptides. INTRODUCTION The enzyme PA catalyzes the hydrolysis of benzyl- penicillin to give phenylacetic acid and 6-APA, the latter being a key intermediate in the production of semisynthetic penicillins (Lowe et al., 1981). The active form of the enzyme consists of two different * To whom correspondence and reprint requests should be addressed, at Apartado Postal 70479, Mi-xico 04510 D.F. (Mexico). Abbreviations: aa, amino acid(s); CAPA, &amino penicillanic acid; bp, base pair(s); DMBA, p-dimethylaminobenzaldehyde; kb, 1000 bp; LB, Luria broth: nt, nucleotidejs); ORF, open reading frame: P4, penicillin acylase; pat, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA E. m/i gene coding for PA: SD, Shine-Dal~rno. subunits of 20.5 and 69 kDa1 (Bock et al., 1983a). These peptides are apparently produced from a precursor peptide, after proteolytic cleavage (Bock et al., 1983b). For many eukaryotic enzymes and hormones, one or more internal proteolytic cleavages are required for full activity (Steiner, 1976). To our knowledge, however, this mechanism has only been described in prokaryotes in the case of PA from ~~c~er~c~~~ cnii ATCClllO.S (Bock et al., 1983a,b). To understand better this novel processing mecha- nism at the molecular level, we have cloned the pat gene and determined the nt sequence of a region that codes for the 20.5kDa1 subunit, as well as the first 78 N-terminal aa of the 69-kDa1 subunit. The pos- sible existence of a connecting peptide between both subunits and the processing steps involved in the formation of the enzyme, are discussed. 037X-1 1 19i85/503.30 0 1985 Elsevicr Scicncc Publishers