PURIFICATION AND CHARACTERIZATION OF EPID, A FLAVOPROTEIN INVOLVED IN THE BIOSYNTHESIS OF THE LANTIBIOTIC EPIDERMIN
PURIFICATION AND CHARACTERIZATION OF EPID, A FLAVOPROTEIN INVOLVED IN THE BIOSYNTHESIS OF THE LANTIBIOTIC EPIDERMIN
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DOI：
10.1128/jb.174.16.5354-5361.1992
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发表时间：
1992-08-01
影响因子：
3.2
通讯作者：
GOTZ, F
中科院分区：
文献类型：
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作者：
KUPKE, T;STEVANOVIC, S;GOTZ, F
利用雄性融合系统和T7RNA聚合酶-启动子系统，在大肠杆菌中表达了表皮葡萄球菌Tu3298的表皮素生物合成基因ependD。用抗麦芽糖结合蛋白(MBP)-EpiD抗血清进行Western blotting(免疫印迹)鉴定EpiD。分别从大肠杆菌克隆中纯化了EpiD和MBP-EpiD融合蛋白，它们主要存在于可溶性蛋白质组分中。纯化的EpiD呈现典型的氧化黄素蛋白的吸收光谱，其最大吸收波长分别为274、382和453 nm。EpiD经热处理后释放的辅酶被鉴定为黄素单核苷酸。含有突变的表皮葡萄球菌Tu3298/EMS11不能合成具有活性的表皮蛋白。该突变基因EPID*在大肠杆菌中被克隆，并以MBP-EpiD*融合蛋白的形式表达。EepD*的DNA测序发现了一个点突变，导致Gly-93被天冬氨酸取代。与MBP-EpiD不同，融合蛋白MBP-EpiD*不能与黄素单核苷酸结合。我们认为，EpiD催化从C端的Meso-lanthionine的半胱氨酸残基上除去两个还原的等价物，形成a-C=C-双键，从而参与了表皮蛋白中不寻常的S-[(Z)-2-氨基乙烯基]-D-半胱氨酸结构的形成。
The plasmid-encoded epidermin biosynthesis gene, epiD, of Staphylococcus epidermidis Tu3298 was expressed in Escherichia coli by using both the malE fusion system and the T7 RNA polymerase-promoter system. EpiD was identified by Western blotting (immunoblotting) with anti-maltose-binding protein (MBP)-EpiD antiserum. EpiD and the MBP-EpiD fusion protein, which were mainly present in the soluble protein fraction, were purified from the respective E. coli clones. Purified EpiD showed the typical absorption spectrum of an oxidized flavoprotein with maxima at 274, 382, and 453 nm. The coenzyme released from EpiD by heat treatment was identified as flavin mononucleotide. S. epidermidis Tu3298/EMS11, containing a mutation within epiD, was unable to synthesize active epidermin. This mutated gene, epiD*, was cloned in E. coli and expressed as an MBP-EpiD* fusion protein. DNA sequencing of epiD* identified a point mutation that led to replacement of Gly-93 with Asp. Unlike MBP-EpiD, the fusion protein MBP-EpiD* could not bind flavin mononucleotide. We propose that EpiD catalyzes the removal of two reducing equivalents from the cysteine residue of the C-terminal meso-lanthionine to form a -C=C- double bond and is therefore involved in formation of the unusual S-[(Z)-2-aminovinyl]-D-Cysteine structure in epidermin.