2.0 Å X‐ray structure of the ternary complex of 7,8‐dihydro‐6‐hydroxymethylpterinpyrophosphokinase from <i>Escherichia coli</i> with ATP and a substrate analogue

Stammers, D.K.; Achari, Aniruddha; Somers, D.O.; Bryant, P.K.; Rosemond, J.; Scott, D. H.; Champness, J.N.

doi:10.1016/s0014-5793(99)00860-1

Cited by 48 publications

(69 citation statements)

References 29 publications

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“…1b) was found in the structure of the complex apparently due to the hydrolysis of the nucleotide. As previously predicted (1) and observed (3,5), the adenine base of ADP is positioned in the V-shaped cleft between ␤4 and ␤6 and the ribose and pyrophosphate extend along the valley and across ␤4 and ␤1 (Fig. 1a).…”

Section: Crystal Structure Of Hppk⅐mgadp At 15-å Resolutionsupporting

confidence: 87%

See 1 more Smart Citation

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: Crystal Structure Of Hppk⅐mgadp At 15-å Resolutionsupporting

confidence: 87%

“…The key elements responsible for pulling down the carboxyl group of Asp 95 appear to be the two bound Mg 2ϩ ions. One Mg 2ϩ is coordinated with the ␣-and ␤-phosphoryl groups of ATP, and the other with the ␤-and ␥-phosphoryl groups of ATP and the hydroxyl group of HP (3,5 Fig. 8b), and so is its coordination.…”

Section: Discussionmentioning

confidence: 99%

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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“…However, the 3′-OH group of GDP or GTP is by itself a rather unreactive species that requires activation via deprotonation, enforced by Glu139 and a magnesium ion. Similar activation of hydroxyl groups has been observed in hydroxymethylpterin pyrophosphokinases (HPPKs) (33). Taken together, these observations show that alarmone synthetases position their substrates in a geometry ideal for catalysis.…”

Section: Discussionsupporting

confidence: 74%

Catalytic mechanism and allosteric regulation of an oligomeric (p)ppGpp synthetase by an alarmone

Steinchen

Schuhmacher

Altegoer

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

109

172

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Nucleotide-based second messengers serve in the response of living organisms to environmental changes. In bacteria and plant chloroplasts, guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively named "(p)ppGpp"] act as alarmones that globally reprogram cellular physiology during various stress conditions. Enzymes of the RelA/SpoT homology (RSH) family synthesize (p)ppGpp by transferring pyrophosphate from ATP to GDP or GTP. Little is known about the catalytic mechanism and regulation of alarmone synthesis. It also is unclear whether ppGpp and pppGpp execute different functions. Here, we unravel the mechanism and allosteric regulation of the highly cooperative alarmone synthetase small alarmone synthetase 1 (SAS1) from Bacillus subtilis. We determine that the catalytic pathway of (p)ppGpp synthesis involves a sequentially ordered substrate binding, activation of ATP in a strained conformation, and transfer of pyrophosphate through a nucleophilic substitution (S N 2) reaction. We show that pppGpp-but not ppGpp-positively regulates SAS1 at an allosteric site. Although the physiological significance remains to be elucidated, we establish the structural and mechanistic basis for a biological activity in which ppGpp and pppGpp execute different functional roles. stringent response | (p)ppGpp | hydrogen-deuterium exchange mass spectrometry | alarmone | crystallography T he ability of living organisms to adapt their metabolism to nutrient limitation or environmental changes is of utmost importance to survival. The stringent response is a highly conserved mechanism that enables bacteria (1-3) and plant chloroplasts (4-6) to respond to nutrient (i.e., amino acid) limitations. However, recent work has indicated that guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively named "(p)ppGpp"] also impact other, nonstringent response processes such as virulence (7-9) as well as persister (10, 11) and biofilm formation (12). Realization of the importance of (p)ppGpp has also opened new avenues for the design of antimicrobial agents (13,14).Central to these processes is the synthesis of two alarmones, pppGpp and ppGpp, which globally reprogram the transcription and translation associated with a variety of cellular processes (summarized in refs. 9 and 15) and which also control the elongation of DNA replication (16). Until now, both alarmones have been collectively named "(p)ppGpp," because knowledge of their individual roles remained mysterious. Only recently has a study indicated that either alarmone might execute disparate biological functions (17).Alarmone synthesis is carried out by synthetases of RelA/SpoT homology (RSH) (18) that catalyze the transfer of pyrophosphate (β-, γ-phosphates) from ATP to the ribose 3′-OH of GDP or GTP to synthesize ppGpp or pppGpp, respectively. An in-depth analysis of the catalytic mechanism for this reaction is currently not available. Only one structure describes the GDP-bound state of an RSH synthetase domain, bearing remarkable simil...

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“…The structure of E. coli hydroxymethyldihydropterin diphosphokinase with a variety of ligands has been determined: these include the ternary complex with a substrate analog and a substrate, enzyme-Mg 2ϩ -␣,␤-methylene ATP-hydroxydihydropterin complex (PDB code 1q0n) (164). Additional reports on crystal or solution structures of hydroxymethyldihydropterin diphosphokinase of E. coli and Haemophilus influenzae have been published (165)(166)(167). Structural and mechanistic properties of this enzyme have been previously reviewed (168).…”

Section: Substitution Reactions At ␣- ␤- or ␥-Phosphatesmentioning

confidence: 99%

Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance

Hove‐Jensen

Andersen

Kilstrup

et al. 2017

Microbiol Mol Biol Rev

149

187

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SUMMARY Phosphoribosyl diphosphate (PRPP) is an important intermediate in cellular metabolism. PRPP is synthesized by PRPP synthase, as follows: ribose 5-phosphate + ATP → PRPP + AMP. PRPP is ubiquitously found in living organisms and is used in substitution reactions with the formation of glycosidic bonds. PRPP is utilized in the biosynthesis of purine and pyrimidine nucleotides, the amino acids histidine and tryptophan, the cofactors NAD and tetrahydromethanopterin, arabinosyl monophosphodecaprenol, and certain aminoglycoside antibiotics. The participation of PRPP in each of these metabolic pathways is reviewed. Central to the metabolism of PRPP is PRPP synthase, which has been studied from all kingdoms of life by classical mechanistic procedures. The results of these analyses are unified with recent progress in molecular enzymology and the elucidation of the three-dimensional structures of PRPP synthases from eubacteria, archaea, and humans. The structures and mechanisms of catalysis of the five diphosphoryltransferases are compared, as are those of selected enzymes of diphosphoryl transfer, phosphoryl transfer, and nucleotidyl transfer reactions. PRPP is used as a substrate by a large number phosphoribosyltransferases. The protein structures and reaction mechanisms of these phosphoribosyltransferases vary and demonstrate the versatility of PRPP as an intermediate in cellular physiology. PRPP synthases appear to have originated from a phosphoribosyltransferase during evolution, as demonstrated by phylogenetic analysis. PRPP, furthermore, is an effector molecule of purine and pyrimidine nucleotide biosynthesis, either by binding to PurR or PyrR regulatory proteins or as an allosteric activator of carbamoylphosphate synthetase. Genetic analyses have disclosed a number of mutants altered in the PRPP synthase-specifying genes in humans as well as bacterial species.

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2.0 Å X‐ray structure of the ternary complex of 7,8‐dihydro‐6‐hydroxymethylpterinpyrophosphokinase from Escherichia coli with ATP and a substrate analogue

Cited by 48 publications

References 29 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

Catalytic mechanism and allosteric regulation of an oligomeric (p)ppGpp synthetase by an alarmone

Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance

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