Wang W Y, Liaw S H, Liao T H
Institute of Biochemistry, College of Medicine, Taiwan University, 1, Sec. 1, Jen-Ai Road, Taipei, Taiwan.
Biochem J. 2000 Mar 15;346 Pt 3(Pt 3):799-804.
Approximately 95% of the amino acid sequence of a shrimp (Penaeus japonicus) nuclease was derived from protease-digested peptides. A 1461-base cDNA for the nuclease was amplified and sequenced with degenerate primers based on the amino acid sequence and then specific primers by 3' and 5' RACE (rapid amplification of cDNA ends). It contains an open reading frame encoding a putative 21-residue signal peptide and a 381-residue mature protein. The N-terminus of the enzyme is pyroglutamate, deduced from composition and matrix-assisted laser desorption ionization-time-of-flight MS analyses, and confirmed by a glutamine residue in the cDNA sequence. The enzyme has 11 Cys residues, forming five intramolecular disulphides. The eleventh Cys residue was linked to a thiol compound with an estimated molecular mass of between 500 and 700 Da. A sequence similarity search revealed no homologous proteins but residues 205-255 shared a conserved active-site motif within a distinct group of nucleases. His(211) in this conserved motif was shown to be very important in catalysis by site-specific modification with (14)C-labelled iodoacetate. The shrimp nuclease, previously designated DNase I, does indeed possess a low level of hydrolytic activity towards RNA in the presence of Mg(2+) and Ca(2+). The conservation of functionally important residues during distant evolution might imply that the catalytic mechanisms are similar in these nucleases, which should be classified in one subfamily. Finally, an active-site structure for shrimp nuclease was proposed on the basis of published structural data and the results of mutational and biochemical analyses of Serratia nuclease.
虾（日本对虾）核酸酶约95%的氨基酸序列来源于蛋白酶消化产生的肽段。基于氨基酸序列设计简并引物，通过3'和5' RACE（cDNA末端快速扩增）技术扩增并测序了该核酸酶的1461个碱基的cDNA。它包含一个开放阅读框，编码一个推测的21个残基的信号肽和一个381个残基的成熟蛋白。通过组成分析和基质辅助激光解吸电离飞行时间质谱分析推断该酶的N端为焦谷氨酸，并通过cDNA序列中的谷氨酰胺残基得到证实。该酶有11个半胱氨酸残基，形成5个分子内二硫键。第11个半胱氨酸残基与一种估计分子量在500至700 Da之间的硫醇化合物相连。序列相似性搜索未发现同源蛋白，但205 - 255位残基在一组独特的核酸酶中共享一个保守的活性位点基序。通过用（14）C标记的碘乙酸进行位点特异性修饰表明，这个保守基序中的His（211）在催化中非常重要。先前被命名为DNase I的虾核酸酶在Mg（2+）和Ca（2+）存在下确实对RNA具有低水平的水解活性。在远距离进化过程中功能重要残基的保守性可能意味着这些核酸酶的催化机制相似，应归为一个亚家族。最后，基于已发表的结构数据以及粘质沙雷氏菌核酸酶的突变和生化分析结果，提出了虾核酸酶的活性位点结构。
