Characterization of a recombinant type II 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Helicobacter pylori.
DAH7P (3-Deoxy-D-arabino-heptulosonate 7-phosphate) synthase catalyses the condensation reaction between phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P) as the first committed step in the biosynthesis of aromatic compounds in plants and micro-organisms. Previous work has identified two families of DAH7P synthases based on sequence similarity and molecular mass, with the majority of the mechanistic and structural studies being carried out on the type I paralogues from Escherichia coli. Whereas a number of organisms possess genes encoding both type I and type II DAH7P synthases, the pathogen Helicobacter pylori has only a single, type II, enzyme. Recombinant DAH7P synthase from H. pylori was partially solubilized by co-expression with chaperonins GroEL/GroES in E. coli, and purified to homogeneity. The enzyme reaction follows an ordered sequential mechanism with the following kinetic parameters: K(m) (PEP), 3 microM; K(m) (E4P), 6 microM; and kcat, 3.3 s(-1). The enzyme reaction involves interaction of the si face of PEP with the re face of E4P. H. pylori DAH7P synthase is not inhibited by phenylalanine, tyrosine, tryptophan or chorismate. EDTA inactivates the enzyme, and activity is restored by a range of bivalent metal ions, including (in order of decreasing effectiveness) Co2+, Mn2+, Ca2+, Mg2+, Cu2+ and Zn2+. Analysis of type II DAH7P synthase sequences reveals several highly conserved motifs, and comparison with the type I enzymes suggests that catalysis by these two enzyme types occurs on a similar active-site scaffold and that the two DAH7P synthase families may indeed be distantly related.
Webby
Celia J
CJ
Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand.
Patchett
Mark L
ML
Parker
Emily J
EJ
D010723:3-Deoxy-7-Phosphoheptulonate Synthase; D000446:Aldehyde-Lyases / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D018833:Chaperonins / Q000378:metabolism; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology*; D016480:Helicobacter pylori / Q000201:enzymology*; D007700:Kinetics; D030781:Organisms, Genetically Modified; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D010723:3-Deoxy-7-Phosphoheptulonate Synthase; D018833:Chaperonins; D000446:Aldehyde-Lyases