Structure and function of poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis T1.
Poly(3-hydroxybutyrate) (PHB) depolymerase from Alcaligenes faecalis T1 is composed of three domains: the catalytic (C) domain, the fibronectin type III-like (F) domain, and the substrate-binding (S) domain. We constructed domain deletion, inversion, chimera, and extra-F-domain mutants and examined their enzyme activity and PHB-binding ability. In addition, we performed substitution of 214Asp and 273His with glycine and aspartate, respectively, to examine their participation in a catalytic triad together with 139Ser. The mutant with both the F and S domains deleted and the trypsin-digested enzyme showed no PHB-hydrolyzing activity and less PHB-binding ability than that of the wild-type enzyme but retained D-(-)-3-hydroxybutyrate trimer-hydrolyzing activity at a level similar to that of the wild-type enzyme. The mutant with the F domain deleted and the mutant which had the order of the F and S domains inverted retained PHB-binding ability and trimer-hydrolyzing activity at levels similar to those of the wild-type enzyme but lost PHB-hydrolyzing activity. The chimera mutant, in which the F domain was substituted with a Thr-rich domain of PHB depolymerase A from Pseudomonas lemoignei, and the extra-F-domain mutant, with an additional F domain, retained trimer- and PHB-hydrolyzing activities and PHB-binding ability at levels similar to those of the wild-type enzyme. Two mutants (D214G and H273D) showed no enzymatic activity toward trimer and PHB, and they were not labeled with [3H]diisopropylfluorophosphate.
Nojiri
M
M
Department of Biological Sciences, Faculty of Science, Kanagawa University, Hiratsuka, Japan.
Saito
T
T
D000421:Alcaligenes / Q000201:enzymology* / Q000235:genetics*; D001216:Asparagine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D007446:Chromosome Inversion; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D015870:Gene Expression; D005998:Glycine / Q000235:genetics; D006639:Histidine / Q000235:genetics; D010957:Plasmids; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000502:physiology; D017384:Sequence Deletion; D012694:Serine / Q000235:genetics; D014357:Trypsin / Q000378:metabolism
D017931:DNA Primers; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D001224:Aspartic Acid; D012694:Serine; D006639:Histidine; D001216:Asparagine; D002265:Carboxylic Ester Hydrolases; C022417:poly-beta-hydroxybutyrate depolymerase; D014357:Trypsin; D005998:Glycine