Catalytic and Anticatalytic Snapshots of a Short-Form ATP Phosphoribosyltransferase

Alphey, M.S.; Fisher, Gemma; Gould, Eoin R.; Machado, Teresa F. G.; Liu, Huanting; Florence, Gordon J.; Naismith, James H.; Silva, Rafael G. da

doi:10.1021/acscatal.8b00867

Cited by 13 publications

(111 citation statements)

References 48 publications

(183 reference statements)

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“…We recently published the crystal structures of Pa HisG S and Pa ATPPRT in binary complexes with PRPP and PRATP, and in ternary complexes with PRPP-ATP, but were unable to obtain structures of enzyme-ATP binary complexes, suggesting a reverse order of substrate binding in comparison with HisG L ATPPRTs. 29 …”

Section: Resultsmentioning

confidence: 99%

“… 11 , 16 , 35 AMP is also a competitive inhibitor against PRPP in L. lactis ATPPRT, 28 and the recent crystal structure of the Pa HisG S -AMP complex shows a similar binding mode as in HisG L ATPPRTs. 11 , 16 , 29 , 35 AMP inhibits Pa HisG S with an IC 50 of 79 ± 6 μM ( Figure S3A ), and inhibition is competitive against both PRPP and ATP, with K i ’s of 25 ± 5 and 52 ± 8 μM, respectively ( Figure S3B,C ). These values are on average ca.…”

Section: Resultsmentioning

confidence: 99%

“… 19 However, crystal structures of Pa HisG S and Pa ATPPRT in complex with PRPP-ADP reveal that ADP binds in the same manner as ATP. 29 In order to evaluate the ability of Pa HisG S to use ADP as a substrate, we compared the reactions with ADP and ATP by 31 P NMR spectroscopy ( Figure S4 ). The spectra of reactions containing ADP ( Figure S4A ) and ATP ( Figure S4C ) are similar except for the peak at −19.2 to −19.4 corresponding to the γ-PO 4 2– phosphorus of ATP and PRATP, since this group is absent in ADP and N 1 -(5-phospho-β- d -ribosyl)-ADP (PRADP).…”

Section: Resultsmentioning

confidence: 99%

“…Theoretical structures were derived from B3LYP calculations using a 6-31G* basis set with a Lanl2DZ basis set on Mg 2+ and Mn 2+ and a Lanl2DZ pseudopotential added to Mg 2+ and Mn 2+ as implemented in Gaussian 09. 31 A model system was chosen by including all residues within 5 Å of ADP and PRPP in the crystallographic dimer of the Pa HisG S -PRPP-Mg-ADP complex crystal structure 29 and by flipping the adenine ring from its crystal structure orientation to bring N1 in proximity to PRPP. The system was further paired down to include only functional groups, metal ions and water molecules within the 5-Å cutoff that were essential for stabilization of the transition structure.…”

Section: Methodsmentioning

confidence: 99%

“… 20 , 24 , 26 , 28 The kinetic mechanism of HisG S ATPPRTs has not been investigated, but recent crystal structures suggest that the order of substrate binding may be different from HisG L ’s. 29 Moreover, little is known about the kinetics of allosteric activation.…”

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Allosteric Activation Shifts the Rate-Limiting Step in a Short-Form ATP Phosphoribosyltransferase

et al. 2018

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Short-form ATP phosphoribosyltransferase (ATPPRT) is a hetero-octameric allosteric enzyme comprising four catalytic subunits (HisGS) and four regulatory subunits (HisZ). ATPPRT catalyzes the Mg2+-dependent condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate (PRPP) to generate N1-(5-phospho-β-d-ribosyl)-ATP (PRATP) and pyrophosphate, the first reaction of histidine biosynthesis. While HisGS is catalytically active on its own, its activity is allosterically enhanced by HisZ in the absence of histidine. In the presence of histidine, HisZ mediates allosteric inhibition of ATPPRT. Here, initial velocity patterns, isothermal titration calorimetry, and differential scanning fluorimetry establish a distinct kinetic mechanism for ATPPRT where PRPP is the first substrate to bind. AMP is an inhibitor of HisGS, but steady-state kinetics and 31P NMR spectroscopy demonstrate that ADP is an alternative substrate. Replacement of Mg2+ by Mn2+ enhances catalysis by HisGS but not by the holoenzyme, suggesting different rate-limiting steps for nonactivated and activated enzyme forms. Density functional theory calculations posit an SN2-like transition state stabilized by two equivalents of the metal ion. Natural bond orbital charge analysis points to Mn2+ increasing HisGS reaction rate via more efficient charge stabilization at the transition state. High solvent viscosity increases HisGS’s catalytic rate, but decreases the hetero-octamer’s, indicating that chemistry and product release are rate-limiting for HisGS and ATPPRT, respectively. This is confirmed by pre-steady-state kinetics, with a burst in product formation observed with the hetero-octamer but not with HisGS. These results are consistent with an activation mechanism whereby HisZ binding leads to a more active conformation of HisGS, accelerating chemistry beyond the product release rate.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Allosteric Activation Shifts the Rate-Limiting Step in a Short-Form ATP Phosphoribosyltransferase

et al. 2018

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Antibacterial Activity of Graphdiyne and Graphdiyne Oxide

Zhu

Bai

et al. 2020

Small

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From manufacture to disposal, the interaction of graphdiyne based nanomaterials with living organisms is inevitable and crucial. However, the cytotoxic properties of this novel carbon nanomaterial are rarely investigated, and the mechanisms behind their cytotoxicity are totally unknown. In this study, the antibacterial activity of graphdiyne (GDY) and graphdiyne oxide (GDYO) is reported. GDY is capable of inhibiting broad‐spectrum bacterial growth while exerting moderate cytotoxicity on mammalian cells. In comparison, GDYO exhibits lower antibacterial activity than that of GDY. Then an alterable, synergetic antibacterial mechanism of GDY, involving wrapping bacterial membrane, membrane insertion and disruption, and reactive oxygen species generation is demonstrated, while the differential gene expression analysis indicates that GDY could only alter the bacterial metabolism slightly and the oxidative stress route may be a minor bactericidal factor. The investigation of the antibacterial behaviors of GDY based nanomaterials may provide useful guidelines for the future design and application of this novel molecular allotrope of carbon.

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Hinge Twists and Population Shifts Deliver Regulated Catalysis for ATP-PRT in Histidine Biosynthesis

Jiao

Mittelstädt

Moggré

et al. 2019

Biophysical Journal

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Allosteric regulation plays an important role in the control of metabolic flux in biosynthetic pathways. In microorganisms, many enzymes in these pathways adopt different strategies of allostery to allow the tuning of their activities in response to metabolic demand. Thus, it is important to uncover the mechanism of allosteric signal transmission to fully comprehend the complex control of enzyme function and its evolution. ATP-phosphoribosyltransferase (ATP-PRT), as the first enzyme in the histidine biosynthetic pathway, is allosterically regulated by histidine and offers a good platform for the study of allostery. Two forms of ATP-PRT, namely long and short forms, were discovered that show different arrangements of their regulatory machinery. Crystal structures of the long-form ATP-PRT have revealed overall conformational changes in the inhibited state, but the observed changes in the active state are quite subtle, making the elucidation of its allosteric mechanism difficult. Here, we combine computational methods (ligand docking, quantum mechanics/molecular mechanics optimization, and molecular dynamic simulations) with experimental studies to probe the signal transmission between remote allosteric and active sites. Our results reveal that distinct conformational ensembles of the catalytic domain with different dynamic properties exist in the ligand-free and histidine-bound enzymes. These ensembles display different capabilities in supporting the catalytic and allosteric function of ATP-PRT. The findings give insight into the underlying mechanism of allostery and allow us to propose that the hinge twisting within the catalytic domain is the key for both enhancement of catalysis and provision of regulation in ATP-PRT enzymes.

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Catalytic and Anticatalytic Snapshots of a Short-Form ATP Phosphoribosyltransferase

Cited by 13 publications

References 48 publications

Allosteric Activation Shifts the Rate-Limiting Step in a Short-Form ATP Phosphoribosyltransferase

Allosteric Activation Shifts the Rate-Limiting Step in a Short-Form ATP Phosphoribosyltransferase

Antibacterial Activity of Graphdiyne and Graphdiyne Oxide

Hinge Twists and Population Shifts Deliver Regulated Catalysis for ATP-PRT in Histidine Biosynthesis

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