Biosynthesis Pathway of ADP- l - glycero -β- d - manno -Heptose in Escherichia coli

Abstract

ABSTRACT The steps involved in the biosynthesis of the ADP- l - glycero -β- d - manno- heptose (ADP- l -β- d -heptose) precursor of the inner core lipopolysaccharide (LPS) have not been completely elucidated. In this work, we have purified the enzymes involved in catalyzing the intermediate steps leading to the synthesis of ADP- d -β- d -heptose and have biochemically characterized the reaction products by high-performance anion-exchange chromatography. We have also constructed a deletion in a novel gene, gmhB (formerly yaeD ), which results in the formation of an altered LPS core. This mutation confirms that the GmhB protein is required for the formation of ADP- d -β- d -heptose. Our results demonstrate that the synthesis of ADP- d -β- d -heptose in Escherichia coli requires three proteins, GmhA (sedoheptulose 7-phosphate isomerase), HldE (bifunctional d -β- d -heptose 7-phosphate kinase/ d -β- d -heptose 1-phosphate adenylyltransferase), and GmhB ( d , d -heptose 1,7-bisphosphate phosphatase), as well as ATP and the ketose phosphate precursor sedoheptulose 7-phosphate. A previously characterized epimerase, formerly named WaaD (RfaD) and now renamed HldD, completes the pathway to form the ADP- l -β- d -heptose precursor utilized in the assembly of inner core LPS.