大肠杆菌青霉素G酰化酶基因及其邻近区域的核苷酸全序列

WHOLE NUCLEOTIDE SEQUENCE OF PENICILLIN G ACYLASE GENE AND ITS FLANKING REGION FROM E.COLI

我们由E.coli AS1.76克隆了青霉素G酰化酶的基因,并且测定了其全部核苷酸序列。青霉素G酰化酶结构基因是由下述功能片段组成的:(1)编码信号肽(26个氨基酸残基)的78个碱基对;(2)编码α-亚基(209个氨基酸残基)的627个碱基对;(3)编码间隔肽(54个氨基酸残基)的162个碱基对;(4)编码β亚基(557个氨基酸残基)的1671个碱基对。此外,我们还发现起始密码子(ATG)前有个核糖体结合位点和启动子序列以及在终止密码子(TAA)之后有个转录终止信号。与最近发表的青霉素G酰化酶基因的DNA序列比较,同源性达99.7%。

Some of microorganisms have been knownto possess penicillin G acylase activity.The E.coli derived penicillin G acylase(PGA) can catalyze the conversion of peni-cillin G into phenylacetic acid and 6-amino-penicillanic acid,the latter is used as thestarting compound for the industrial forma-tion of semi-synthetic penicillins.Apart fromits industrial importance,the enzyme PGAdisplays a number of interesting properties.Catalytically active enzyme is localized inthe periplasmic space of E.coli cells andcomposed of two dissimilar subunits.Thetwo subunits are apparently produced from aprecursor protein,via a processing pathwayhitherto unique in its features for a prokary-otic enzyme.The studies on processing ofthe precursor and on the relationship betweenstructure and function of the mature enzymeare important theoretically. Previously we cloned a 3.5 kb DNAfragment from a strain (E.coli AS 1.76),which displays PGA activity.In this paper,we report a nucleotide sequence of the 3.5kb DNA fragment containing PGA gene.After insertion of the DNA fragment intoEcoR Ⅰ and Hind Ⅲ sites in pWR 13,pPGA 20 had been obtained.We subclonedthe Hind Ⅲ and Bgl Ⅱ treated fragment of1.6 kb in length from pPGA 20 into HindⅢ and BamH Ⅰ sites of pWR 13 to get apPGA 1.6.and Bgl Ⅱ and EcoR Ⅰ treatedfragment of 1.9 kb in length into BamH Ⅰand EcoR Ⅰ sites of pWR 13 to get a pPGA1.9.The linearized pPGA 1.9 which weredigested with appropriate restriction enzymeswere progressively shortened from both endsrespectively by digestion with Bal 31 nu-clease,followed by cleavage of shortened tar-get DNA off vector DNA molcules with ap-proriate restriction enzymes.The series ofthe DNA fragments shortened from EcoR Ⅰend were then cloned into plasmid pWR 13which had previously digested with Hind Ⅲand Sma Ⅰ enzymes (Fig.1).The DNA frag-ment cloned in pWR 13 were directly se-quenced on the resulted plasmids by usingprimer Ⅰ and primer Ⅱ.Thus we have ob-tained the complete nucleotide sequence ofthe 3.5 kb DNA fragment. The 3.5 kb fragment contains an in-tact PGA gene which is 2.6 kb.The openreading frame in the gene consists of fourstructural domains:(i) nucleotide positionsNo.53—130 coding for a signal peptide of theprecusor enzyme,(ii) positions No.131—757coding for alpha-subunit of the mature en-zyme,(iii) positions No.758—919 coding forthe spacer peptide of the precusor enzyme,(iv) positions No.920—2590 coding for beta-subunit of the mature enzyme.Additionallyupstream from the starting codon ATG,thereis a promoter and a PBS (i,e.SD) sequen-ce,and downstream sequence from termina-tion codon TAA,there is a terminator.(Fig.3).