CHARACTERIZATION OF SACCHAROPOLYSPORA-ERYTHRAEA CYTOCHROME-P-450 GENES AND ENZYMES, INCLUDING 6-DEOXYERYTHRONOLIDE-B HYDROXYLASE
CHARACTERIZATION OF SACCHAROPOLYSPORA-ERYTHRAEA CYTOCHROME-P-450 GENES AND ENZYMES, INCLUDING 6-DEOXYERYTHRONOLIDE-B HYDROXYLASE
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DOI：
10.1128/jb.174.3.725-735.1992
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发表时间：
1992-02-01
影响因子：
3.2
通讯作者：
HUTCHINSON, CR
中科院分区：
文献类型：
--
作者：
ANDERSEN, JF;HUTCHINSON, CR
先前的红霉素生物合成研究表明，细胞色素P-450单加氧酶系统负责6-脱氧红素内酯B羟基化为红素内酯B，作为红霉素合成的一部分（a . Shafiee和C. R. Hutchinson, Biochemistry 26:6204-6210 1987）。该酶先前被纯化到明显的均匀性，并发现催化周转数约为10(-3)min-1。最近，在红链球菌的红霉素耐药基因ermE区域的p -450编码序列（eryF）被破坏，产生了一个6-脱氧红素内酯B羟化缺陷突变体（J. M. Weber， J. O. Leung， S. J. Swanson， K. B. Idler和J. B. McAlpine, Science, 252:114-116, 1991）。本研究从赤藓中纯化了具有催化活性的细胞色素P-450组分，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳发现其包含一个主要的P-450组分和一个次要的P-450组分。从基因组DNA中克隆了主要种（orf405）的编码基因，发现与eryF不同。orf405和eryF基因均在大肠杆菌中表达，并比较了两者蛋白的性质。异源表达的EryF和Orf405均与Shafiee和Hutchinson（1987）描述的6-脱氧红素内酯B羟化酶制备的抗血清发生反应，并且EryF多肽在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳凝胶上与S. erythraea的小P-450种发生反应。在酶活性的比较中，EryF羟基化了一个周转数为53 min-1的底物，而Orf405对6-脱氧红素B类似物没有检测到活性。这两种酶在7-乙氧基香豆素的o -脱烷基反应中均表现出较弱的活性。我们得出结论，先前分离的6-脱氧红素B羟化酶是两种P-450酶的混合物，只有次要形式显示6-脱氧红素B羟化酶活性。
Previous studies of erythromycin biosynthesis have indicated that a cytochrome P-450 monooxygenase system is responsible for hydroxylation of 6-deoxyerythronolide B to erythronolide B as part of erythromycin biosynthesis in Saccharopolyspora erythraea (A. Shafiee and C. R. Hutchinson, Biochemistry 26:6204-6210 1987). The enzyme was previously purified to apparent homogeneity and found to have a catalytic turnover number of approximately 10(-3) min-1. More recently, disruption of a P-450-encoding sequence (eryF) in the region of ermE, the erythromycin resistance gene of S. erythraea, produced a 6-deoxyerythronolide B hydroxylation-deficient mutant (J. M. Weber, J. O. Leung, S. J. Swanson, K. B. Idler, and J. B. McAlpine, Science 252:114-116, 1991). In this study we purified the catalytically active cytochrome P-450 fraction from S. erythraea and found by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis that it consists of a major and a minor P-450 species. The gene encoding the major species (orf405) was cloned from genomic DNA and found to be distinct from eryF. Both the orf405 and eryF genes were expressed in Escherichia coli, and the properties of the proteins were compared. Heterologously expressed EryF and Orf405 both reacted with antisera prepared against the 6-deoxyerythronolide B hydroxylase described by Shafiee and Hutchinson (1987), and the EryF polypeptide comigrated with the minor P-450 species from S. erythraea on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. In comparisons of enzymatic activity, EryF hydroxylated a substrate with a turnover number of 53 min-1, whereas Orf405 showed no detectable activity with a 6-deoxyerythronolide B analog. Both enzymes showed weak activity in the O-dealkylation of 7-ethoxycoumarin. We conclude that the previously isolated 6-deoxyerythronolide B hydroxylase was a mixture of two P-450 enzymes and that only the minor form shows 6-deoxyerythronolide B hydroxylase activity.