Cloning and characterization of a novel 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Salvia miltiorrhiza involved in diterpenoid tanshinone accumulation.
The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate (MVA), which is a rate-limiting step in the isoprenoid biosynthesis via the MVA pathway. In this study, the full-length cDNA encoding HMGR (designated as SmHMGR2, GenBank accession no. FJ747636) was isolated from Salvia miltiorrhiza by rapid amplification of cDNA ends (RACE). The cloned gene was then transformed into the hairy root of S. miltiorrhiza, and the enzyme activity and production of diterpenoid tanshinones and squalene were monitored. The full-length cDNA of SmHMGR2 comprises 1959bp, with a 1653-bp open reading frame encoding a 550-amino-acid protein. Molecular modeling showed that SmHMGR2 is a new HMGR with a spatial structure similar to other plant HMGRs. SmHMGR2 contains two HMG-CoA-binding motifs and two NADP(H)-binding motifs. The SmHMGR2 catalytic domain can form a homodimer. The deduced protein has an isoelectric point of 6.28 and a calculated molecular weight of approximately 58.67kDa. Sequence comparison analysis showed that SmHMGR2 had the highest homology to HMGR from Atractylodes lancea. As expected, a phylogenetic tree analysis indicates that SmHMGR2 belongs to plant HMGR group. Tissue expression pattern analysis shows that SmHMGR2 is strongly expressed in the leaves, stem, and roots. Functional complementation of SmHMGR2 in HMGR-deficient mutant yeast JRY2394 demonstrates that SmHMGR2 mediates the MVA biosynthesis in yeasts. Overexpression of SmHMGR2 increased enzyme activity and enhanced the production of tanshinones and squalene in cultured hairy roots of S. miltiorrhiza. Our DNA gel blot analysis has confirmed the presence and integration of the associated SmHMGR2 gene. SmHMGR2 is a novel and important enzyme involved in the biosynthesis of diterpenoid tanshinones in S. miltiorrhiza.
Dai
Zhubo
Z
Institute of Chinese Materia Medica, Chinese Academy of Chinese Medical Science, Beijing, China.
Cui
Guanghong
G
Zhou
Shu-Feng
SF
Zhang
Xianan
X
Huang
Luqi
L
D045784:Abietanes; D000214:Acyl Coenzyme A / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019295:Computational Biology; D004224:Diterpenes / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010616:Phenanthrenes / Q000378:metabolism*; D010802:Phylogeny; D018517:Plant Roots / Q000201:enzymology / Q000235:genetics; D016133:Polymerase Chain Reaction; D027561:Salvia miltiorrhiza / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D045784:Abietanes; D000214:Acyl Coenzyme A; D004224:Diterpenes; D010616:Phenanthrenes; C021751:tanshinone; C008047:3-hydroxy-3-methylglutaryl-coenzyme A