Essential role of residue H49 for activity of Escherichia coli 1-deoxy-D-xylulose 5-phosphate synthase, the enzyme catalyzing the first step of the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid Synthesis.
The first step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in plant plastids and most eubacteria is catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase (DXS), a recently described transketolase-like enzyme. To identify key residues for DXS activity, we compared the amino acid sequence of Escherichia coli DXS with that of E. coli and yeast transketolase (TK). Alignment showed a previously undetected conserved region containing an invariant histidine residue that has been described to participate in proton transfer during TK catalysis. The possible role of the conserved residue in E. coli DXS (H49) was examined by site-directed mutagenesis. Replacement of this histidine residue with glutamine yielded a mutant DXS-H49Q enzyme that showed no detectable DXS activity. These findings are consistent with those obtained for yeast TK and demonstrate a key role of H49 for DXS activity.
Querol
J
J
Departament de Bioquímica i Biologia Molecular, Universitat de Barcelona, Martí i Franquès 1-7, 08028 Barcelona, Spain.
Rodríguez-Concepción
M
M
Boronat
A
A
Imperial
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D017124:Conserved Sequence; D017931:DNA Primers / Q000235:genetics; D004896:Erythritol / Q000031:analogs & derivatives* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006639:Histidine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011105:Polyisoprenyl Phosphate Sugars / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013403:Sugar Phosphates / Q000378:metabolism*; D014166:Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014174:Transketolase / Q000235:genetics
C114232:2-C-methylerythritol 4-phosphate; D017931:DNA Primers; D011105:Polyisoprenyl Phosphate Sugars; D013403:Sugar Phosphates; D006639:Histidine; D014166:Transferases; C109461:deoxyxylulose-5-phosphate synthase; D014174:Transketolase; D004896:Erythritol