Identification of a short (C15) chain Z-isoprenyl diphosphate synthase and a homologous long (C50) chain isoprenyl diphosphate synthase in Mycobacterium tuberculosis.
We report the cloning, overexpression, and partial characterization of two unique Z-isoprenyl diphosphate synthase homologs from Mycobacterium tuberculosis. The first enzyme, Rv1086, adds one isoprene unit to omega,E-geranyl diphosphate. The product, omega,E, Z-farnesyl diphosphate, is the putative substrate of the second enzyme, Rv2361c. This enzyme adds seven more isoprene units to omega, E,Z-farnesyl diphosphate and releases decaprenyl diphosphate. Both open reading frames were cloned from the M. tuberculosis H37Rv genome and overexpressed in M. smegmatis. Membrane and cytosol fractions from wild type and the two recombinant strains were assayed for [(14)C]isopentenyl diphosphate incorporation into isoprenyl diphosphates in the presence of various allylic isoprenyl diphosphate acceptors. Membrane fractions of recombinant cells overexpressing Rv2361c incubated with farnesyl diphosphate showed a 10-fold increase of [(14)C]isopentenyl diphosphate incorporation into decaprenyl diphosphate. Membrane fractions of recombinant cells overexpressing Rv1086 incubated with geranyl diphosphate showed a 5-fold increase of [(14)C]isopentenyl diphosphate incorporation into farnesyl diphosphate. Analysis of the stereochemistry revealed that all of the overexpressed farnesyl diphosphate was in the omega,E, Z-configuration. This is the first description of a short chain isoprenyl diphosphate synthase that generates products with Z-stereochemistry. Previously, all known short chain isoprenyl diphosphate synthases catalyze the synthesis of products with E-stereochemistry.
Schulbach
M C
MC
Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523, USA.
Brennan
P J
PJ
Crick
D C
DC
D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D002384:Catalysis; D002855:Chromatography, Thin Layer; D017931:DNA Primers; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012717:Sesquiterpenes
D001426:Bacterial Proteins; D017931:DNA Primers; D011106:Polyisoprenyl Phosphates; D011994:Recombinant Proteins; D012717:Sesquiterpenes; C004808:farnesyl pyrophosphate; D019883:Alkyl and Aryl Transferases; C412569:Rv1086 protein, Mycobacterium tuberculosis; C499606:Rv2361c protein, Mycobacterium tuberculosis