Poly-3-hydroxybutyrate synthase from the periplasm of Escherichia coli.
This is the first report of a poly-3-hydroxybutyrate (PHB) synthase in Escherichia coli. The enzyme was isolated from the periplasm using ammonium sulfate fractionation, hydrophobic, and size-exclusion chromatography and identified by LC/MS/MS as YdcS, a component of a putative ABC transporter. Green Fluorescent Protein-tagged ydcS, purified by 2D native gel electrophoresis, also exhibited PHB synthase activity. Optimal conditions for enzyme activity were 37 degrees C, pH 6.8-7.5, 100 mM KCl. K(m) was 0.14 mM and V(max) was 18.7 nmol/mg protein/min. The periplasms of deletion mutants displayed <25% of the activity of the parent strain. Deletion mutants exhibited approximately 25% less growth in M9 medium, glucose, and contained approximately 30% less PHB complexed to proteins (cPHB) in the outer membranes, but the same concentration of chloroform-extractable PHB as wild-type cells. The primary sequence of YdcS suggests it may belong to the alpha-/beta-hydrolase superfamily which includes polyhydroxybutyrate (PHB) synthases, lipases, and esterases.
Dai
Dongsheng
D
Department of Microbiology and Molecular Genetics, Michigan State University, 6163 Biomedical Biophysical Science, East Lansing, MI 48824, USA.
Reusch
Rosetta N
RN
D000217:Acyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D002725:Chloroform / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017353:Gene Deletion; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D019897:Periplasm / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D002725:Chloroform; D000217:Acyltransferases; C531117:YdcS protein, E coli; C060970:poly-beta-hydroxybutyrate polymerase