Molecular characterization of hasB from an operon required for hyaluronic acid synthesis in group A streptococci. Demonstration of UDP-glucose dehydrogenase activity.
The membrane-associated hyaluronate synthase produces capsular hyaluronate in group A streptococci by the alternate addition of UDP-N-acetylglucosamine and UDP-glucuronic acid. Previous studies identified a locus required for hyaluronate synthase activity and suggested that a gene involved in the production of UDP-glucuronic acid (UDP-glucose dehydrogenase) also mapped to the locus. In the present study the putative UDP-glucose dehydrogenase gene (hasB) was cloned and the DNA sequence determined. The hasB gene product was shown to have global similarity with AlgD, a dehydrogenase, which catalyzes the production of GDP-mannuronic acid for the alginate capsule of Pseudomonas aeruginosa. Regions of local homology have been identified which apparently correspond to the NAD-binding and enzyme active sites of HasB and AlgD. In order to show that hasB expression correlated with UDP-glucose dehydrogenase activity, the hasB gene was cloned under control of the T7 promoter. Hyperexpression of hasB resulted in a protein of approximately 47 kDa and high levels of UDP-glucose dehydrogenase activity were observed. These data demonstrate that hasB encodes the UDP-glucose dehydrogenase of group A streptococci.
Dougherty
B A
BA
Wake Forest University Medical Center, Winston-Salem, North Carolina 27157.
van de Rijn
I
I
D000595:Amino Acid Sequence; D001483:Base Sequence; D002237:Carbohydrate Dehydrogenases / Q000235:genetics; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006820:Hyaluronic Acid / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009876:Operon; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013291:Streptococcus / Q000201:enzymology / Q000235:genetics*; D014533:Uridine Diphosphate Glucose Dehydrogenase / Q000235:genetics* / Q000378:metabolism
D004269:DNA, Bacterial; D006820:Hyaluronic Acid; D002237:Carbohydrate Dehydrogenases; C020562:GDPmannose dehydrogenase; D014533:Uridine Diphosphate Glucose Dehydrogenase