Equilibrium and Kinetic Studies of Substrate Binding to 6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase fromEscherichia coli

Bermingham, Alun; Bottomley, Joanna; Primrose, William U.; Derrick, Jeremy P.

doi:10.1074/jbc.m000331200

Cited by 39 publications

(83 citation statements)

References 25 publications

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“…The crystal structure of HPPK⅐MgADP and the solution structure of HPPK⅐MgAMPPCP may represent the intermediate conformations required for assembling the catalytic center. The idea is consistent with the ordered bi-bi mechanism suggested by Bermingham and co-workers (21 (Fig. 8b).…”

Section: Discussionsupporting

confidence: 79%

Unusual Conformational Changes in 6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase as Revealed by X-ray Crystallography and NMR

Xiao

Shi²,

Gao³

et al. 2001

Journal of Biological Chemistry

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Because the folate pathway is essential for microorganisms but absent from mammals, HPPK, like other enzymes in the pathway, is an important target for developing antimicrobial agents.HPPK belongs to a class of enzymes that catalyze the pyrophosphoryl transfer reaction (2). Although the mechanisms of many kinases that catalyze monophosphoryl transfer have been extensively characterized, little is known about the mechanisms of pyrophosphokinases. As a small (158 residues, ϳ18 kDa), stable, monomeric protein, Escherichia coli HPPK is an excellent model system for studying the mechanisms of enzymatic pyrophosphoryl transfer.We have recently determined the crystal structures of ligand-free (apo-) E. coli HPPK (1) and its complex with HP, ␣,␤-methyleneadenosine 5Ј-triphosphate (AMPCPP), and two Mg 2ϩ ions (3) at 1.5 and 1.25 Å resolution, respectively. Hennig and co-workers (4) have determined the crystal structure of Haemophilus influenzae HPPK in complex with an HP analog at 2.05-Å resolution. Stammers and co-workers (5) have determined the crystal structure of E. coli HPPK in complex with an HP analog, ATP, and two Mg 2ϩ ions at 2.00-Å resolution. Comparative analysis of the crystal structures of the apo-HPPK and its ternary complex has revealed the interactions of the enzyme with the substrates at the atomic resolution and the dramatic substrate-induced conformational changes involving three catalytic loops (3). It appears that the complete active center of HPPK is assembled only after both substrates bind to the enzyme. However, how the catalytic center is assembled is not known. It appears that some catalytic residues cannot move into their catalytic positions because of steric constraints.In this paper, we present the crystal structure of HPPK⅐MgADP at 1.5-Å resolution and the NMR solution structure of HPPK⅐MgAMPPCP. The two structures reveal a dramatic, unusual movement of loop 3 and other significant changes in the conformation and dynamical property of HPPK. The dramatic substrate-induced movement of loop 3 is unusual because it moves away from the active center. Comparative structural analysis suggests that the structures reported here may represent an intermediate conformation required for both substrate binding and product release in the catalytic cycle. EXPERIMENTAL PROCEDURESCrystal Structure Determination of HPPK⅐MgADP-E. coli HPPK was purified as previously described (6). Crystals of HPPK⅐MgADP were grown in hanging drops at 19 Ϯ 1°C. The protein solution contained 4 mg/ml HPPK, 5 mM MgATP in 10 mM Tris buffer (pH 8.0), and the reservoir contained 30% polyethylene glycol 4000, 0.2 M NaAc in 0.2 M Tris buffer (pH 8.5). The 4-l hanging drops contained equal volumes (2 l) of protein solution and reservoir solution. Two rounds of seeding produced diffraction-quality crystals. * This work was supported in part by National Institutes of Health Grant GM51901 (to H. Y.). This study made use of a Varian INOVA-600 NMR spectrometer at Michigan State University funded in part by National Science Foundation Grant...

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Section: Discussionsupporting

confidence: 79%

Unusual Conformational Changes in 6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase as Revealed by X-ray Crystallography and NMR

Xiao

Shi²,

Gao³

et al. 2001

Journal of Biological Chemistry

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“…A variety of crystallographic and enzyme studies on Escherichia coli HPPK have identified the binding sites for the two substrates, ATP and 6-hydroxymethyl-7,8-dihydropterin (HMDP). 5,[8][9][10][11][12][13][14] Three loop regions are involved in a structural change, induced on binding of ATP, to form the binding site for HMDP: this primes the active site for the direct transfer of the pyrophosphoryl moiety from ATP to HMDP.…”

Section: Introductionmentioning

confidence: 99%

Crystal Structure of the Bifunctional Dihydroneopterin Aldolase/6-hydroxymethyl-7,8-dihydropterin Pyrophosphokinase from Streptococcus pneumoniae

Garçon

Levy

Derrick

2006

Journal of Molecular Biology

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“…The inward movement of this loop appears to be responsible both for the formation of the active site upon the binding of the substrate MgATP and for the trapping of the product 6-hydroxymethyl-7,8-dihydropterin pyrophosphate upon completion of the reaction. The binding of MgATP likely occurs first,42,43 as HPPK has only a low affinity for 6-hydroxymethyl-7,8-dihydropterin in the absence of MgATP. Whilst loop 3 is largely disordered in our structure, three lines of reasoning suggest that the (disordered) conformations spanned by this loop are of an "open" form, i.e.…”

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confidence: 99%

The Three-dimensional Structure of the Bifunctional 6-Hydroxymethyl-7,8-Dihydropterin Pyrophosphokinase/Dihydropteroate Synthase of Saccharomyces cerevisiae

Lawrence

Iliades

Fernley

et al. 2005

Journal of Molecular Biology

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Equilibrium and Kinetic Studies of Substrate Binding to 6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase fromEscherichia coli

Cited by 39 publications

References 25 publications

Unusual Conformational Changes in 6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase as Revealed by X-ray Crystallography and NMR

Unusual Conformational Changes in 6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase as Revealed by X-ray Crystallography and NMR

Crystal Structure of the Bifunctional Dihydroneopterin Aldolase/6-hydroxymethyl-7,8-dihydropterin Pyrophosphokinase from Streptococcus pneumoniae

The Three-dimensional Structure of the Bifunctional 6-Hydroxymethyl-7,8-Dihydropterin Pyrophosphokinase/Dihydropteroate Synthase of Saccharomyces cerevisiae

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