An Alkaline D-Stereospecific Endopeptidase with β-Lactamase Activity from Bacillus cereus*
An Alkaline D-Stereospecific Endopeptidase with β-Lactamase Activity from Bacillus cereus*
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来自蜡样芽孢杆菌的具有 β-内酰胺酶活性的碱性 D-立体特异性内肽酶*
DOI：
10.1074/jbc.271.47.30256
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Y. Kato
中科院分区：
文献类型：
--
作者：
Y. Asano;H. Ito;T. Dairi;Y. Kato
我们从土壤细菌蜡状芽孢杆菌DF4-B菌株的培养液中纯化了一种新的胞外D-立体特异性内肽酶--碱性D-肽酶(D-立体特异性多肽水解酶，EC 3.4.11-)。该酶的相对分子质量为37,952，由单一的多肽链组成。最适pH为∼10.3。该酶对(D-Phe)_3、(D-Phe)_4等由D苯丙氨酸组成的寡肽具有严格的D-立体特异性，对(D-Phe)_6、Boc-(D-Phe)_4(其中Boc为叔丁氧基)、Boc-(D-Phe)_4甲酯、Boc-(D-Phe)_3甲酯、Boc-(D-Phe)_2、(D-Phe)_2等不起作用，但对由L-Phe、(D-Ala)n(n=2-5)组成的多肽不起作用。用合成底物((D-Phe)2-D-Tyr和D-Tyr-(D-Phe)2)和(Phe)3的8个立体异构体明确了该酶的作用方式。该酶对氨苄西林和青霉素G有β-内酰胺酶活性，但羧肽酶DD和D-氨基肽酶活性不明显。将编码碱性D-肽酶(ADP)的基因克隆到pUC118载体上，经鉴定为ADP基因，全长1164个碱基，388个密码子。预测的多肽与链霉菌R61的羧肽酶DD、链霉菌的青霉素结合蛋白和枯草杆菌的青霉素结合蛋白以及C类β-内酰胺酶相似。因此，该酶被归类为一种新的“青霉素识别酶”。
We purified a novel extracellular D-stereospecific endopeptidase, alkaline D-peptidase (D-stereospecific peptide hydrolase, EC 3.4.11.-), to homogeneity from the culture broth of the soil bacterium Bacillus cereus strain DF4-B. The Mr of the enzyme was 37,952, and it was composed of a single polypeptide chain. The optimal pH for activity was ∼10.3. The enzyme was strictly D-stereospecific toward oligopeptides composed of Dphenylalanine such as (D-Phe)3 and (D-Phe)4. The enzyme also acted to a lesser extent on (D-Phe)6, Boc-(D-Phe)4 (where Boc is tert-butoxycarbonyl), Boc-(D-Phe)4 methyl ester, Boc-(D-Phe)3 methyl ester, Boc-(D-Phe)2, (D-Phe)2, and others, but not upon their corresponding peptides composed of L-Phe, (D-Ala)n (n = 2-5), (D-Val)3, and (D-Leu)2. The mode of action of the enzyme was clarified with synthetic substrates ((D-Phe)2-D-Tyr and D-Tyr-(D-Phe)2) and eight stereoisomers of (Phe)3. The enzyme had β-lactamase activity toward ampicillin and penicillin G, although carboxypeptidase DD and D-aminopeptidase activities were undetectable. The gene coding for alkaline D-peptidase (adp) was cloned into plasmid pUC118, and a 1164-base pair open reading frame consisting of 388 codons was identified as the adp gene. The predicted polypeptide was similar to carboxypeptidase DD from Streptomyces R61, penicillin-binding proteins from Streptomyces lactamdurans and Bacillus subtilis, and class C β-lactamases. Thus, the enzyme was categorized as a new “penicillin-recognizing enzyme.”