Cloning, expression, and characterization of the Escherichia coli K-12 rfaD gene

Abstract

The rfaD gene encodes ADP-L-glycero-D-mannoheptose-6-epimerase, an enzyme required for the biosynthesis of the lipopolysaccharide precursor ADP-L-glycerol-D-mannoheptose. The precise localization of the rfaD gene on a 1.3-kilobase SspI-HpaI fragment is reported. The rfaD gene and the flanking regions were completely sequenced. The location of the rfaD gene on the physical map of the Escherichia coli chromosome was determined. Primer extension studies were used to define the regulatory region of the rfaD gene. The cloned rfaD gene directed the synthesis of a 37,000-dalton polypeptide in several in vivo and in vitro expression systems. N-terminal analysis of purified ADP-L-glycero-D-mannoheptose-6-epimerase confirmed the first 34-amino-acid sequence deduced from the nucleotide sequence of the rfaD gene coding region. The primary structure of the rfaD protein contains the sequence fingerprint for the ADP-binding beta alpha beta fold at the N terminus.