THE DHNA GENE OF ESCHERICHIA-COLI ENCODES A CLASS-I FRUCTOSE BISPHOSPHATE ALDOLASE

The gene encoding the Escherichia coli Class I fructose-1,6-bisphospha te aldolase (FBP aldolase) has been cloned and the protein overproduce d in high amounts. This gene sequence has previously been identified a s encoding an E. coli dehydrin in the GenBank(TM) database [gene dhnA; entry code U73760; Close and Choi (1996) Submission to GenBank(TM)]. However, the purified protein overproduced from the dhnA gene shares a ll its properties with those known for the E. coli Class I FBP aldolas e. The protein is an 8-10-mer with a native molecular mass of approx. 340 kDa, each subunit consisting of 349 amino acids. The Class I enzym e shows low sequence identity with other known FBP aldolases, both Cla ss I and Class II (in the order of 20%), which may be reflected by som e novel properties of this FBP aldolase. The active-site peptide has b een isolated and the Schiff-base-forming lysine residue (Lys(236)) has been identified by a combination of site-directed mutagenesis, kineti cs and electrospray-ionization MS. A second lysine residue (Lys(238)) has been implicated in substrate binding. The cloning of this gene and the high levels of overexpression obtained will facilitate future str ucture function studies.