Mosquito glucosamine-6-phosphate N-acetyltransferase: cDNA, gene structure and enzyme kinetics

Mosquito midgut epithelial cells secrete digestive enzymes as well as components of the peritrophic matrix in response to blood-feeding. The peritrophic matrix is composed of proteins, glycoproteins and chitin fibrils in a proteoglycan matrix and may function to protect the midgut epithelium from mechanical damage and insult from pathogens and toxins. Chitin biosynthesis takes place via the hexosamine pathway converting fructose-6-phsophate to UDP-N-acetylglucosamine, which is then polymerized to chitin by chitin synthase. Glucosamine-6-phosphate N-acetyltransferase (GNA) is one of the hexosamine pathway enzymes and catalyzes the transfer of the acetyl group from acetyl-CoA to the primary amine of glucosamine-6-phosphate. We cloned and sequenced the GNA cDNA, gene (AeGna) and its putative promoter regions from Aedes aegypti. AeGna consists of five exons and four introns and lacks a TATA box near the transcription start site. The AeGna cDNA is 1.3 kb in length and the predicted protein is approximately 23.6 kDa. The amino acid sequence of AeGna has high homology to its orthologues. AeGna mRNA is constitutively expressed in all developmental stages and blood-feeding causes no obvious effect on levels of AeGna transcript in the midgut. The Km value of recombinant GNA for glucosamine-6-phosphate was 330 μM and the Km for acetyl-CoA was 500 μM.