Molecular Modeling, Cloning And Over-expression Of Homologous Deoxyriboaldolases

Eight gram positive strains were chosen via genome mining, and the putative 2-Deoxy-D-Ribose-5-phosphate aldolases (DERA) genes, BamDERA, GthDERA, SepDERA, BthDERA, LmoDERA, BsuDERA, CaceDERA, BliDERA, origined from the chromosome of these microorganisms were individually cloned via PCR and over-expressed in Escherichia coli. It was found that these gram positive DERAs had high tolerance towards heat, considering 40-100% of the activity could be retained when they were heated at 70℃, especially GthDERA remained 77% of the activity when it was heated at 100℃. On the other hand, E. coli DERA(EcoDERA) was largely inactivated by incubation at temperatures of 60℃. They had similar cleavage activity for their natural substrate 2-deoxy-D-ribose-5-phosphate(DRP), compared with EcoDERA, while they exhibited higher catalysis ability over EcoDERA in the retroaldol reaction of nonphosphorylated substrate 2-deoxyribose(DR). The ratio of specificity constants for DR compared to DRP ([kcat/Km(DR)]/[kcat/Km(DRP)]) of eight gram positive strains DERAs were all two or three orders of magnitude higher than EcoDERA.Eight structural models of cloned DERAs had been generated through homology modeling. The substrate-binding pocket were predicted based on the alignment and superposition eight DERAs with EcoDERA, It showed similar structure characters that most residues consisted of substrate-binding pocket was conservative, except Lys172, Val206, Arg207 and Ser239 in EcoDERA were replaced by Phe, Ile, His and Ala in CaeDERA; Lys172, Val206 and Ser239 were replaced by Phe, Ile and Ala in BsuDERA, BliDERA, BamDERA; Lys172 and Ser239 were replaced by Phe and Ala in BthDERA, LmoDERA and SepDERA; Lys172 was replaced by Phe in GthDERA, respectively. Phe200 was replaced by Val in eight DERAs.The differences might affect the electrostatic environment in substrate-binding pocket and the hydrophobic interaction around the scaffold of the two key Lys residues, herein were responsible for the differences in substrate specificities. The result of docking DRP and DR to DERA is consistent with result of the experiment, the DR docking score of eight DERAs was one or two orders of magnitude higher than EcoDERA.