Wagemaker Matthijs J M, Eastwood Daniel C, van der Drift Chris, Jetten Mike S M, Burton Kerry, Van Griensven Leo J L D, Op den Camp Huub J M
Department of Microbiology, IWWR, Radboud University Nijmegen, Toernooiveld 1, NL-6525 ED Nijmegen, The Netherlands.
Mycol Res. 2007 Apr;111(Pt 4):493-502. doi: 10.1016/j.mycres.2007.01.016. Epub 2007 Feb 8.
Accumulation of high quantities of urea in fruiting bodies is a known feature of larger basidiomycetes. Argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) are two ornithine cycle enzymes catalysing the last two steps in the arginine biosynthetic pathway. Arginine is the main precursor for urea formation. In this work the nucleotide sequences of the genes and corresponding cDNAs encoding argininosuccinate synthetase (ass) and argininosuccinate lyase (asl) from Agaricus bisporus were determined. Eight and six introns were present in the ass and asl gene, respectively. The location of four introns in the asl gene were conserved among vertebrate asl genes. Deduced amino acid sequences, representing the first homobasidiomycete ASS and ASL protein sequences, were analysed and compared with their counterparts in other organisms. The ass ORF encoded for a protein of 425 amino acids with a calculated molecular mass of 47266Da. An alignment with ASS proteins from other organisms revealed high similarity with fungal and mammalian ASS proteins, 61-63% and 51-55% identity, respectively. The asl open reading frame (ORF) encoded a protein of 464 amino acids with an calculated mass of 52337Da and similar to ASS shared the highest similarity with fungal ASL proteins, 59-60% identity. Northern analyses of ass and asl during fruiting body formation and post-harvest development revealed that expression was significantly up-regulated from developmental stage 3 on for all the tissues studied. The expression reached a maximum at the later stages of fruiting body growth, stages 6 and 7. Both ass and asl genes were up-regulated within 3h after harvest showing that the induction mechanism is very sensitive to the harvest event and emphasizes the importance of the arginine biosynthetic pathway/ornithine cycle in post-harvest physiology.
在子实体中积累大量尿素是大型担子菌的一个已知特征。精氨琥珀酸合成酶（ASS）和精氨琥珀酸裂解酶（ASL）是鸟氨酸循环中的两种酶，催化精氨酸生物合成途径的最后两步。精氨酸是尿素形成的主要前体。在这项工作中，测定了双孢蘑菇中编码精氨琥珀酸合成酶（ass）和精氨琥珀酸裂解酶（asl）的基因及相应cDNA的核苷酸序列。ass基因和asl基因分别存在8个和6个内含子。asl基因中4个内含子的位置在脊椎动物asl基因中是保守的。推导的氨基酸序列代表了首个同担子菌的ASS和ASL蛋白序列，并与其他生物体中的对应序列进行了分析和比较。ass开放阅读框编码一个425个氨基酸的蛋白质，计算分子量为47266Da。与其他生物体的ASS蛋白比对显示，与真菌和哺乳动物的ASS蛋白高度相似，同一性分别为61 - 63%和51 - 55%。asl开放阅读框（ORF）编码一个464个氨基酸的蛋白质，计算分子量为52337Da，与ASS类似，与真菌ASL蛋白的相似性最高，同一性为59 - 60%。对子实体形成和采后发育过程中ass和asl的Northern分析表明，在所研究的所有组织中，从发育阶段3开始表达显著上调。在子实体生长的后期阶段6和7表达达到最大值。ass和asl基因在收获后3小时内均上调，表明诱导机制对收获事件非常敏感，并强调了精氨酸生物合成途径/鸟氨酸循环在采后生理学中的重要性。
