A Point Mutation Converts Dihydroneopterin Aldolase to a Cofactor-Independent Oxygenase

Wang, Yi; Scherperel, Gwynyth; Roberts, Kade D.; Jones, A. Daniel; Reid, Gavin E.; Yan, Honggao

doi:10.1021/ja063455i

Cited by 13 publications

(29 citation statements)

References 24 publications

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“…Y54 can be excluded on the basis of our previous site-directed mutagenesis of the conserved tyrosine residue (13). The present site-directed mutagenesis study suggests that both E22 and K100 are important for catalysis, with K100 contributing a bit more to the transition state stabilization.…”

Section: Discussionmentioning

confidence: 66%

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

Wang

Yan

2006

Biochemistry

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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. ...

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Section: Discussionmentioning

confidence: 66%

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

Wang

Yan

2006

Biochemistry

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“…11,12 The carboxyl group of E22 is hydrogen bonded to the ε-amino group of K100 and the 1′-hydroxyl group in all three complexes, whereas Y54′ and K100 interact with the ligand differently ( Figure 4). The ε-amino group of K100 and the hydroxyl group of Y54′ are hydrogen bonded to the 2′-hydroxyl of NP (Figure 4(a)), but to the 3′-hydroxyl of MP ( Figure 4(b)).…”

Section: Four Snapshots Along the Catalytic Pathwaymentioning

confidence: 99%

“…9 An active site lysine residue (K100) has been proposed to function as the general base and a bound water as a proton donor, leading to the first reaction scheme predicted for DHNA. 6 Recently, our biochemical and biophysical studies have yielded further insights into the mechanism of DHNA, including the important roles of active site glutamate (E22) and tyrosine (Y54) residues, 11,12 and the reversible nature of DHNA-catalyzed epimerization reaction (Y. Wang et al, unpublished results). Here, we present two crystal structures that make it possible to derive the critical interactions between DHNA and the trihydroxypropyl moiety of the substrate, providing further structural insights into the mechanism of DHNA-catalyzed reactions.…”

Section: Introductionmentioning

confidence: 99%

Structural Basis for the Aldolase and Epimerase Activities of Staphylococcus aureus Dihydroneopterin Aldolase

Błaszczyk

Gan

et al. 2007

Journal of Molecular Biology

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Dihydroneopterin aldolase (DHNA) catalyzes the conversion of 7,8-dihydroneopterin (DHNP) to 6-hydroxymethyl-7,8-dihydropterin (HP) and the epimerization of DHNP to 7,8-dihydromonopterin (DHMP). Although crystal structures of the enzyme from several microorganisms have been reported, no structural information is available about the critical interactions between DHNA and the trihydroxypropyl moiety of the substrate, which undergoes bond cleavage and formation. Here, we present the structures of Staphylococcus aureus DHNA (SaDHNA) in complex with neopterin (NP, an analog of DHNP) and with monapterin (MP, an analog of DHMP), filling the gap in the structural analysis of the enzyme. In combination with previously reported SaDHNA structures in its ligand-free form (PDB entry 1DHN) and in complex with HP (PDB entry 2DHN), four snapshots for the catalytic center assembly along the reaction pathway can be derived, advancing our knowledge about the molecular mechanism of SaDHNA-catalyzed reactions. An additional step appears to be necessary for the epimerization of DHMP to DHNP. Three active site residues (E22, K100, and Y54) function coordinately during catalysis: together, they organize the catalytic center assembly, and individually, each plays a central role at different stages of the catalytic cycle.

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“…In the structure of bacterial DHNAs, a conserved tyrosine residue (Y54 in S. aureus DHNA) appears at almost the same position as Y111 in M. jannaschii DHNA (Fig. 5), and it has been proposed to act as a catalytic acid in the protonation of the enol intermediate (30). A point mutation of this tyrosine converts DHNA into an oxygenase (30).…”

Section: Discussionmentioning

confidence: 99%

“…5), and it has been proposed to act as a catalytic acid in the protonation of the enol intermediate (30). A point mutation of this tyrosine converts DHNA into an oxygenase (30). This tyrosine could also be modulating water accessibility to the active site, which is required to protonate the N5 of DHNP (27).…”

Section: Discussionmentioning

confidence: 99%

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

Wang

Grochowski

et al. 2014

J Bacteriol

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). Archaeal DHNA shows a unique secondary and quaternary structure compared with bacterial and plant DHNAs. Here, we report a detailed biochemical examination of DHNA from the methanogen Methanocaldococcus jannaschii. Kinetic studies show that M. jannaschii DHNA possesses a catalytic capability with a k cat /K m above 10 5 M ؊1 s ؊1 at 70°C, and at room temperature it exhibits a turnover number (0.07 s ؊1 ) comparable to bacterial DHNAs. We also found that this enzyme follows an acid-base catalytic mechanism similar to the bacterial DHNAs, except when using alternative catalytic residues. We propose that in the absence of lysine, which is considered to be the general base in bacterial DHNAs, an invariant water molecule likely functions as the catalytic base, and the strictly conserved His35 and Gln61 residues serve as the hydrogen bond partners to adjust the basicity of the water molecule. Indeed, substitution of either His35 or Gln61 causes a 20-fold decrease in k cat . An invariant Tyr78 is also shown to be important for catalysis, likely functioning as a general acid. Glu25 plays an important role in substrate binding, since replacing Glu25 by Gln caused a >25-fold increase in K m . These results provide important insights into the catalytic mechanism of archaeal DHNAs.

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A Point Mutation Converts Dihydroneopterin Aldolase to a Cofactor-Independent Oxygenase

Cited by 13 publications

References 24 publications

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

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

Structural Basis for the Aldolase and Epimerase Activities of Staphylococcus aureus Dihydroneopterin Aldolase

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

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