Sirinupong Nualpun, Suwanmanee Pluang, Doolittle Russell F, Suvachitanont Wallie
Biochemistry Department, Faculty of Science, Prince of Songkla University, Hat Yai, 90112, Thailand.
Planta. 2005 Jun;221(4):502-12. doi: 10.1007/s00425-004-1463-7. Epub 2005 Mar 3.
3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS), EC 4.1.3.5, is an essential enzyme in rubber biosynthesis in Hevea brasiliensis. We have isolated a new cDNA encoding HMGS in H. brasiliensis. The full-length hmgs2 consists of 1,916-bp and encodes a protein of 464 amino acids with a predicted molecular mass of 51.27 kDa and an isoelectric point of 6.02. In comparison, HMGS1 and HMGS2 show 92% and 94% nucleotide and amino acid sequence identities, respectively. Semiquantitative RT-PCR analysis indicates that the hmgs2 is more highly expressed in laticifer and petiole than in leaves. Sequence searching and alignment revealed that HMGS is a distant relative of the condensing enzyme; beta-ketoacyl acyl carrier protein synthase III (ACP synthase III), EC 2.3.1.41, identified three completely conserved residues; Cys(117), His(247), and Asn(326). The relationship was greatly strengthened by making a proper alignment of numerous sequences of both HMGS and ACP synthase III. The same Cys(117), His(247), and Asn(326) absolutely conserved in both groups play a catalytic role in ACP synthase III, while such a role of Cys and His has only been reported for HMGS. According to site-directed mutagenesis, the expressed wild-type enzyme shows comparable level with mutant proteins. The mutation of Cys(117) and Asn(326) affects the HMGS activity, indicating that Cys(117) and Asn(326) are important amino acids for the catalytic activity of HMGS. A phylogenetic tree constructed on the basis of proper multiple alignment indicates that HMGS1 and HMGS2 result from recent gene duplication. This is also the case for HMGS and ACP synthase III, which appear to have arisen from an ancient gene duplication event of an ancestral condensing enzyme. Therefore, a possible secondary structure of HMGS could be predicted based on the Protein Data Bank information of ACP synthase III.
3-羟基-3-甲基戊二酰辅酶A合酶（HMGS），酶编号EC 4.1.3.5，是巴西橡胶树橡胶生物合成中的一种关键酶。我们已从巴西橡胶树中分离出一个编码HMGS的新cDNA。hmgs2全长1916个碱基对，编码一个由464个氨基酸组成的蛋白质，预测分子量为51.27 kDa，等电点为6.02。相比之下，HMGS1和HMGS2的核苷酸和氨基酸序列同一性分别为92%和94%。半定量RT-PCR分析表明，hmgs2在乳管和叶柄中的表达高于叶片。序列搜索和比对显示，HMGS是缩合酶β-酮酰基酰基载体蛋白合酶III（ACP合酶III，酶编号EC 2.3.1.41）的远亲；鉴定出三个完全保守的残基：半胱氨酸（Cys）（117）、组氨酸（His）（247）和天冬酰胺（Asn）（326）。通过对HMGS和ACP合酶III的众多序列进行适当比对极大地加强了这种关系。两组中完全保守的相同的半胱氨酸（Cys）（117）、组氨酸（His）（247）和天冬酰胺（Asn）（326）在ACP合酶III中起催化作用，而半胱氨酸和组氨酸在HMGS中的这种作用仅有报道。根据定点诱变，表达的野生型酶与突变蛋白显示出相当的水平。半胱氨酸（Cys）（117）和天冬酰胺（Asn）（326）的突变影响HMGS活性，表明半胱氨酸（Cys）（117）和天冬酰胺（Asn）（326）是HMGS催化活性的重要氨基酸。基于适当的多重比对构建的系统发育树表明，HMGS1和HMGS2是近期基因复制的结果。HMGS和ACP合酶III也是如此，它们似乎源于一个祖先缩合酶的古老基因复制事件。因此，可根据ACP合酶III的蛋白质数据库信息预测HMGS可能的二级结构。
