Structure and function relationship of dihydroneopterin aldolases from Escherichia coli and Staphylococcus aureus

Dihydroneopterin aldolase (DHNA) catalyzes the cleavage of 7,8-dihydro-D-neopterin (DHNP) to form 6-hydroxymethyl-7,8-dihydropterin (HP) and glycolaldehyde and the epimerization of DHNP to form 7,8-dihydro-L-monapterin (DHMP). NMR analysis of the reaction products in a D2O solvent suggests that the epimerization reaction follows the same intermediate as the aldol reaction. A complete set of kinetic constants for both the aldol and epimerization reactions according to a unified kinetic mechanism has been determined for DHNA from Staphylococcus aureus (SaDHNA) and DHNA from Escherichia coli (EcDHNA). The results show that they have significantly different binding and catalytic properties, in accordance with the significant sequence differences between them. EcDHNA is different from SaDHNA biochemically in several aspects. EcDHNA has much higher affinities for the substrate, products, and inhibitors measured in this work. EcDHNA has a much higher epimerase activity than SaDHNA. The rate-limiting step is product release for EcDHNA but is the chemical step for SaDHNA. EcDHNA has significantly higher rate constants for the chemical steps than SaDHNA.; The functional role of a conserved tyrosine residue at the active site of DHNA has been investigated by site-directed mutagenesis. Comprehensive analysis of the reactions catalyzed by Y54F-SaDHNA and Y53F-EcDHNA showed that the major reaction product is dihydroxanthopterin (DHXP) rather than HP. DHXP is generated via the same enol intermediate as in the wild-type enzyme-catalyzed reaction. The mutants are impaired in the protonation of the enol intermediate to form HP. In addition to the normal products and DHXP, formic acid is also formed in the reaction. In addition to DHNP, molecular oxygen is also consumed in the reaction. The ligand-binding properties of the mutants are perturbed to a small extent. The results showed that the mutant enzymes are oxygenases, and the conserved tyrosine residue plays only a minor role in the physical steps of the enzymatic reaction and the formation of the enol reaction intermediate but a critical role in the protonation of the enol intermediate to form HP.; The functional roles of the conserved glutamate and lysine residues at the active site, E22, E74, and K100 in SaDHNA, E21, E73, and K98 in EcDHNA, have been investigated by site-directed mutagenesis in this work. The results showed that E74 of SaDHNA and E73 of EcDHNA are important for substrate binding, but their roles in catalysis are minor if any. In contrast, E22 and K100 of SaDHNA are important for catalysis, but their roles in substrate binding are minor. On the other hand, E21 and K98 of EcDHNA are important for both substrate binding and catalysis.