ABSTRACT
The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR; EC1.1.1.34) catalyzes the first committed step of isoprenoids biosynthesis in Mevalonate (MVA) pathway. Here we report for the first time the cloning and characterization of a full-length cDNA encoding HMGR from Fritillaria cirrhosa (FcHMGR), a bulbous medicinal plant. The full-length cDNA of FcHMGR was 2072 base pair (bp), containing a 1680-bp open reading frame. Bioinformatical analyses revealed that FcHMGR had HMG CoA-binding domains and two NADPH binding domains, which are required for HMGR activity. Quantitative real-time PCR (qRT-PCR) analysis revealed that FcHMGR expressed high in mature bulbs. A truncated version of FcHMGR protein lacking the N-terminal 249-bp GC rich area was expressed in Escherichia coli. The crude cell lysate containing the recombinant protein showed a better HMGR activity than the control and the relative enzyme activity was calculated to be 1.62 U/mg. The cloning, characterization and functional analysis of FcHMGR gene allowed us to further understand the role of FcHMGR involved in steroidal alkaloid biosynthetic pathway in F. cirrhosa at the molecular level.
Keywords:
3-Hydroxy-3-methylglutaryl-CoA reductases; Fritillaria cirrhosa; Molecular cloning; Expression pattern; Prokaryotic expression
(The N-terminal truncated region is underlined; The 3' and 5' RACE primers were marked with forward and reverse solid arrows, respectively. Asterisk denotes translation terminator)
(The HMG-CoA binding domains and NADPH binding domains were marked with solid line and dotted line, respectively. The highly conserved regions of the alignments have been shaded)
(A) Phylogenetic tree of FcHMGR protein sequences from different species. (B) FcHMGR protein tertiary structure.
(A) SDS-PAGE (12%) analysis of FcNt-HMGR over-expression in E.coli BL21. Lane M, protein marker (Beyotime p0061); lane 1, without IPTG induction; lane 2, IPTG induction (arrow mark is 75 kDa); lane 3, the supernatant of lysate without IPTG induction; lane 4, the supernatant of lysate with IPTG induction. (B) The enzyme activity assay by measuring the absorbance attenuation of NADPH at 340 nm. CK, non-induced recombinant FcNt-HMGR BL21 cells; IPTG, induced recombinant FcNt-HMGR BL21 cells.