Cloning, expression and characterization of a dipteran farnesyl diphosphate synthase.
Farnesyl diphosphate synthase (FPPS) of the dipteran Drosophila melanogaster has been cloned and its catalytic properties have been assessed. Analysis of the D. melanogaster genome and of ESTs indicates that FPPS is a single copy gene that produces two transcripts, which differ only by 5' extension. The cDNA of shorter and longer D. melanogaster FPPSs (DmFPPS-1a and DmFPPS-1b, respectively) were each subcloned into pET28a and expressed as an N-His6 fusion protein in BL21 E. coli cells. The DmFPPSs similarly catalyzed the coupling of the allylic substrates, GPP and DMAPP, with IPP, producing FPP as product. The longer protein was further characterized. The enzyme required divalent metal for activity, and was activated by 0.1% Triton X-100. Higher detergent concentration and the addition of glycerol, conditions that activate certain insect FPPSs, inhibited prenyl coupling by DmFPPS-1b. Although DmFPPS-1b does not efficiently couple homologous GPP compounds, homodimethylallyl diphosphate (HDMAPP), which is precursor to all homologous JH structures, was a reactive substrate.
Sen
Stephanie E
SE
Department of Chemistry, Indiana U.-Purdue U. Indianapolis (IUPUI), 402 North Blackford Street, Indianapolis, IN 46202, USA. sen@chem.iupui.edu
Trobaugh
Corey
C
Béliveau
Catherine
C
Richard
Thenesha
T
Cusson
Michel
M
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D020224:Expressed Sequence Tags; D018628:Gene Dosage; D051228:Geranyltranstransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity
D029721:Drosophila Proteins; D051228:Geranyltranstransferase