PhaC and PhaR are required for polyhydroxyalkanoic acid synthase activity in Bacillus megaterium.
Polyhydroxyalkanoic acids (PHAs) are a class of polyesters stored in inclusion bodies and found in many bacteria and in some archaea. The terminal step in the synthesis of PHA is catalyzed by PHA synthase. Genes encoding this enzyme have been cloned, and the primary sequence of the protein, PhaC, is deduced from the nucleotide sequences of more than 30 organisms. PHA synthases are grouped into three classes based on substrate range, molecular mass, and whether or not there is a requirement for phaE in addition to the phaC gene product. Here we report the results of an analysis of a PHA synthase that does not fit any of the described classes. This novel PHA synthase from Bacillus megaterium required PhaC (PhaC(Bm)) and PhaR (PhaR(Bm)) for activity in vivo and in vitro. PhaC(Bm) showed greatest similarity to the PhaCs of class III in both size and sequence. Unlike those in class III, the 40-kDa PhaE was not required, and furthermore, the 22-kDa PhaR(Bm) had no obvious homology to PhaE. Previously we showed that PhaC(Bm), and here we show that PhaR(Bm), is localized to inclusion bodies in living cells. We show that two forms of PHA synthase exist, an active form in PHA-accumulating cells and an inactive form in nonaccumulating cells. PhaC was constitutively produced in both cell types but was more susceptible to protease degradation in the latter type. Our data show that the role of PhaR is posttranscriptional and that it functions directly or indirectly with PhaC(Bm) to produce an active PHA synthase.
McCool
G J
GJ
Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, Massachusetts 01003, USA.
Cannon
M C
MC
D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001410:Bacillus megaterium / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D012097:Repressor Proteins; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C118828:PHAP protein, Bacteria; D012097:Repressor Proteins; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase; C060970:poly-beta-hydroxybutyrate polymerase