Beck J, Ripka S, Siegner A, Schiltz E, Schweizer E
Lehrstuhl für Biochemie, Universität Erlangen-Nürnberg, Federal Republic of Germany.
Eur J Biochem. 1990 Sep 11;192(2):487-98. doi: 10.1111/j.1432-1033.1990.tb19252.x.
6-Methylsalicylic acid synthase (MSAS) from Penicillium patulum is a homomultimer of a single, multifunctional protein subunit. The enzyme is induced, at the transcriptional level, during the end of the logarithmic growth phase. After approximately 150-fold purification, a homogeneous enzyme preparation was obtained exhibiting, upon SDS gel electrophoresis, a subunit molecular mass of 188 kDa. By immunological screening of a genomic P. patulum DNA expression library, the MSAS gene together with its flanking sequences was isolated; 7131 base pairs of the cloned genomic DNA were sequenced. Within this sequence the MSAS gene was identified as a 5322-bp-long open reading frame coding for a protein of 1774 amino acids and 190,731 Da molecular mass. Transcriptional initiation and termination sites were determined both by primer extension studies and from cDNA sequences specially prepared for the 5' and 3' portions of the gene. The same cDNA sequences revealed the presence of a 69-bp intron within the N-terminal part of the MSAS gene. The intron contains the canonical GT and AG dinucleotides at its 5'- and 3'-splice junctions. An internal TACTGAC sequence, resembling the TACTAAC consensus element of Saccharomyces cerevisiae introns is suggested to represent the branch point of the lariat splicing intermediate. When compared to other known polyketide synthases, distinct amino acid sequence similarities of limited lengths were observed with some, though not all, of them. A comparatively low degree of similarity was detected to the yeast and Penicillium FAS or to the plant chalcone and resveratrol synthases. In contrast, a significantly higher sequence similarity was found between MSAS and the rat fatty acid synthase, especially at their transacylase, 2-oxoacyl reductase, 2-oxoacyl synthase and acyl carrier protein domains. Besides several dissimilar, interspersed regions probably coding for MSAS- and FAS-specific functions, the sequential order of the similar domains was colinear in both enzymes. The low similarity between the two P. patulum polyketide synthases, MSAS and FAS, possibly supports a convergent rather than a divergent evolution of both multienzyme proteins.
来自展青霉的6-甲基水杨酸合酶（MSAS）是由单一多功能蛋白质亚基组成的同多聚体。该酶在对数生长期末期在转录水平上被诱导。经过约150倍的纯化后，获得了一种均一的酶制剂，在SDS凝胶电泳中显示亚基分子量为188 kDa。通过对展青霉基因组DNA表达文库的免疫筛选，分离出了MSAS基因及其侧翼序列；对克隆的基因组DNA的7131个碱基对进行了测序。在该序列中，MSAS基因被鉴定为一个5322 bp长的开放阅读框，编码一个由1774个氨基酸组成、分子量为190731 Da的蛋白质。通过引物延伸研究以及专门为该基因的5'和3'部分制备的cDNA序列，确定了转录起始和终止位点。相同的cDNA序列显示在MSAS基因的N端部分存在一个69 bp的内含子。该内含子在其5'和3'剪接连接处含有典型的GT和AG二核苷酸。一个内部的TACTGAC序列，类似于酿酒酵母内含子的TACTAAC共有元件，被认为代表套索状剪接中间体的分支点。与其他已知的聚酮合酶相比，在其中一些（并非全部）酶中观察到了有限长度的明显氨基酸序列相似性。与酵母和青霉脂肪酸合酶或植物查尔酮和白藜芦醇合酶相比，检测到的相似性程度相对较低。相比之下，在MSAS和大鼠脂肪酸合酶之间发现了明显更高的序列相似性，特别是在它们的转酰基酶、2-氧代酰基还原酶、2-氧代酰基合酶和酰基载体蛋白结构域。除了几个可能编码MSAS和FAS特异性功能的不同的、散布的区域外，两种酶中相似结构域的顺序是共线的。展青霉的两种聚酮合酶MSAS和FAS之间的低相似性可能支持这两种多酶蛋白的趋同进化而非分歧进化。
