Molecular cloning of a new cDNA and expression of 3-hydroxy-3-methylglutaryl-CoA synthase gene from Hevea brasiliensis.
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.
Sirinupong
Nualpun
N
Biochemistry Department, Faculty of Science, Prince of Songkla University, Hat Yai, 90112, Thailand.
Suwanmanee
Pluang
P
Doolittle
Russell F
RF
Suvachitanont
Wallie
W
D000214:Acyl Coenzyme A / Q000096:biosynthesis; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D015870:Gene Expression; D028482:Hevea / Q000201:enzymology* / Q000235:genetics*; D006904:Hydroxymethylglutaryl-CoA Synthase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D000214:Acyl Coenzyme A; D018076:DNA, Complementary; C008047:3-hydroxy-3-methylglutaryl-coenzyme A; D006904:Hydroxymethylglutaryl-CoA Synthase
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