Mechanism of Dihydroneopterin Aldolase:  Functional Roles of the Conserved Active Site Glutamate and Lysine Residues

Abstract

Dihydroneopterin aldolase (DHNA) catalyzes the conversion of 7,8-dihydroneopterin (DHNP) to in the folate biosynthetic pathway. There are four conserved active site residues at the active site, E22, Y54, E74, and K100 in Staphylococcus aureus DHNA (SaDHNA), corresponding to E21, Y53, E73, and K98 in Escherichia coli DHNA (EcDHNA). The functional roles of the conserved glutamate and lysine residues have been investigated by site-directed mutagenesis in this work. E22 and E74 of SaDHNA and E21, E73, and K98 of EcDHNA were replaced by alanine. K100 of SaDHNA was replaced by alanine and glutamine. The mutant proteins were characterized by equilibrium binding, stopped-flow binding, and steady-state kinetic analyses. For SaDHNA, none of the mutations except E74A caused dramatic changes in the affinities of the enzyme for the substrate or product analogues or the rate constants. The K d values for SaE74A were estimated to be >3000 μM, suggesting that the K d values of the mutant is at least 100 times those of the wild-type enzyme. For EcDHNA, the E73A mutation caused increases in the K d values for the substrate or product analogues neopterin (MP), monapterin (NP), and 6-hydroxypterin (HPO) by factors of 340, 160, and 5600, respectively, relative to those of the wild-type enzyme. The K98A mutation caused increases in the K d values for NP, MP, and HPO by factors of 14, 3.6, and 230, respectively. The E21A mutation caused increases in the K d values for NP and HPO by factors of 2.2 and 42, respectively, but a decrease in the K d value for MP by a factor of 3.3. The E22 (E21) and K100 (K98) mutations caused decreases in the k cat values by factors of 1.3×10 4 to 2×10 4 . The E74 (E73) mutation caused decreases in the k cat values by factors of ~10. The results suggested that E74 of SaDHNA and E73 of EcDHNA are important for substrate binding, but their roles in catalysis are minor. 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.Dihydroneopterin aldolase (DHNA) catalyzes the conversion of the 7,8-dihydroneopterin (DHNP) to 6-hydroxymethyl-7,8-dihydropterin (HP) in the folate biosynthetic pathway, one of principal targets for developing antimicrobial agents (1-3). Folate cofactors are essential for life (4). Most microorganisms must synthesize folates de novo. In contrast, mammals cannot synthesize folates because of the lack of three enzymes in the middle of the folate pathway and obtain folates from the diet. DHNA is the first of the three enzymes that are absent in mammals and therefore an attractive target for developing antimicrobial agents (5).DHNA is a unique aldolase in two respects. First, DHNA requires neither the formation of a Schiff base between the substrate and enzyme nor metal ions for catalysis (6). Aldolases can † This work was supported in part by NIH grant GM51901 (H.Y.).* To whom correspondence should be addressed. yanh@msu.edu. ...

Biochemical Characterization of a Dihydroneopterin Aldolase Used for Methanopterin Biosynthesis in Methanogens

“…The DHNA from M. jannaschii exhibited a turnover number (0.07 s Ϫ1 at 25°C) comparable to that of bacterial DHNAs, which have reported turnover numbers ranging from 0.005 to 0.08 s Ϫ1 . However, its K m is 10-to 500-fold higher than those of bacterial DHNAs (23,27).…”

“…It is reasonable to assume that Glu25 from M. jannaschii DHNA plays a similar role. Replacement of this corresponding glutamic acid in bacterial DHNA causes a significant increase in K m (23). Similarly, the E25Q variant exhibited a K m of ϳ250 M, which is 25-fold higher than that of the wild-type enzyme, further indicating that Glu25 is an anchor for substrate binding.…”

“…DHNAs from bacteria are cofactor and metal independent. As a result, the mechanism for catalyzing the cleavage of a carbon-carbon bond was proposed based on general acid-base catalysis (23,24). DHNA from M. jannaschii is also cofactor and metal independent.…”

“…N5-protonated substrate in the binary complex (15,23). It is reasonable to assume that Glu25 from M. jannaschii DHNA plays a similar role.…”

“…Its proposed mechanism is similar to that seen in the class I enzymes, but the Schiff base is embedded in the substrate. The mechanism of bacterial DHNA has been studied (23,24) and is expected to involve the formation of the enamine intermediate from the imine in the D-7,8-dihydroneopterin (DHNP) substrate. Mutagenesis studies with enzymes from Escherichia coli and Staphylococcus aureus implicated a conserved lysine and glutamate residue as being involved in the catalytic mechanism, with the lysine acting as the catalytic base, which is required to deprotonate the 2=-hydroxyl of DHNP (15,23,24).…”