Kinetics and Structure of a Cold-Adapted Hetero-Octameric ATP Phosphoribosyltransferase

Stroek, Rozanne; Talbot, Paul; Glok, Mateusz K.; Bernaś, Klaudia E.; Thomson, Catherine M.; Gould, Eoin R.; Alphey, M.S.; Liu, Huanting; Florence, Gordon J.; Naismith, James H.; Silva, Rafael G. da

doi:10.1021/acs.biochem.6b01138

Cited by 17 publications

(120 citation statements)

References 49 publications

(175 reference statements)

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“…ESI-MS analysis showed the expected mass of 718.0 (Figure S1A inset), and the 31 P-NMR spectrum ( Figure S1B) displayed chemical shifts identical to those previously assigned. 18 Crystal structures of apo, PRPP-, PRPP-ATP-, and PRATP-bound PaHisGS. To gain insight into catalysis, PaHisGS crystal structures were solved in apo form and in complex with substrates and product.…”

Section: Resultsmentioning

confidence: 99%

“…Subunits in the homodimer interact in a head-to-tail arrangement ( Figure S2A and B) almost identical to the arrangement found in the PaATPPRT structure. 18 SEC-MALS analysis was used to establish the quaternary structure of PaHisGS in solution ( Figure S3), resulting in a calculated molecular mass of ca. 48000, consistent with a homodimer (the monomer mass is 25215).…”

Section: Resultsmentioning

confidence: 99%

“…ATP and ADP library files were generated with AceDRG. 32 The coordinate and library files for PRATP were generated using PRODRG 33 along with other reaction components as previously described, 18 in a Shimadzu UV-2600 spectrophotometer. Measurements were carried out in duplicate.…”

Section: Crystallisation Ofmentioning

confidence: 99%

“…PaATPPRT. It is well known that HisGS catalysis is activated by HisZ, 15,18,20 but the mechanism of activation is still elusive. We have recently published the structure of…”

Section: Crystal Structures Of Prpp- Prpp-atp- Pratp- and Prpp-adpmentioning

confidence: 99%

“…[16][17] HisGS is found in complex with HisZ, a catalytically inactive regulatory protein, the product of the hisZ gene, a paralog of histidyl-tRNA synthetase, forming the ATPPRT holoenzyme, a hetero-octamer where a HisZ tetramer is flanked by two HisGS dimers. [17][18][19] HisZ has a dual function: it allosterically enhances catalysis by HisGS, and it binds histidine and therefore mediates allosteric inhibition. 17,20 Synthetic biology efforts have attempted to harness the histidine biosynthetic pathway for industrial production of histidine in bacteria, [21][22][23] as engineering of amino acid biosynthesis in microbes is a valuable tool for biocatalytic industrial production of these compounds.…”

Section: Introductionmentioning

confidence: 99%

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Catalytic and Anticatalytic Snapshots of a Short-Form ATP Phosphoribosyltransferase

Alphey¹,

Fisher²,

Gould³

et al. 2018

ACS Catal.

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101

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Allosteric modulation of catalysis is a common regulatory strategy of flux-controlling biosynthetic enzymes. The enzyme ATP phosphoribosyltransferase (ATPPRT) catalyses the first reaction in histidine biosynthesis, the magnesium-dependent condensation of ATP and 5phospho--D-ribosyl-1-pyrophosphate (PRPP) to generate N 1-(5-phospho--D-ribosyl)-ATP (PRATP) and pyrophosphate (PPi). ATPPRT is allosterically inhibited by the final product of the pathway, histidine. Hetero-octameric ATPPRT consists of four catalytic subunits (HisGS) and four regulatory subunits (HisZ) engaged in intricate catalytic regulation. HisZ enhances HisGS catalysis in the absence of histidine while mediating allosteric inhibition in its presence. Here we report HisGS structures for the apoenzyme and complexes with substrates (PRPP, PRPP-ATP, PRPP-ADP), product (PRATP), and inhibitor (AMP), along with ATPPRT holoenzyme structures in complexes with substrates (PRPP, PRPP-ATP, PRPP-ADP) and product (PRATP). These ten crystal structures provide an atomic view of the catalytic cycle and allosteric activation of Psychrobacter arcticus ATPPRT. In both ternary complexes with PRPP-ATP, the adenine ring is found in an anticatalytic orientation, rotated 180° from the catalytic rotamer. Arg32 interacts with phosphate groups of ATP and PRPP, bringing the substrates in proximity for catalysis. The negative charge repulsion is further attenuated by a magnesium ion sandwiched between the and -phosphate groups of both substrates. HisZ binding to form the hetero-octamer brings HisGS subunits closer together in a tighter dimer in the Michaelis complex, which poises Arg56 from the adjacent HisGS molecule for crosssubunit stabilisation of the PPi leaving group at the transition state. The more electrostatically pre-organised active site of the holoenzyme likely minimises the reorganisation energy required to accommodate the transition state. This provides a structural basis for allosteric activation in which chemistry is accelerated by facilitating leaving group departure.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Crystallisation Ofmentioning

confidence: 99%

“…PaATPPRT. It is well known that HisGS catalysis is activated by HisZ, 15,18,20 but the mechanism of activation is still elusive. We have recently published the structure of…”

Section: Crystal Structures Of Prpp- Prpp-atp- Pratp- and Prpp-adpmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Catalytic and Anticatalytic Snapshots of a Short-Form ATP Phosphoribosyltransferase

Alphey¹,

Fisher²,

Gould³

et al. 2018

ACS Catal.

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101

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Human 2′‐Deoxynucleoside 5′‐Phosphate N‐Hydrolase 1: The Catalytic Roles of Tyr24 and Asp80

Carberry,

Devi,

Harrison

et al. 2024

ChemBioChem

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The human enzyme 2′‐deoxynucleoside 5′‐phosphate N‐hydrolase 1 (HsDNPH1) catalyses the hydrolysis of 5‐hydroxymethyl‐2′‐deoxyuridine 5′‐phosphate to generate 5‐hydroxymethyluracil and 2‐deoxyribose‐5‐phosphate via a covalent 5‐phospho‐2‐deoxyribosylated enzyme intermediate. HsDNPH1 is a promising target for inhibitor development towards anticancer drugs. Here, site‐directed mutagenesis of conserved active‐site residues, followed by HPLC analysis of the reaction and steady‐state kinetics are employed to reveal the importance of each of these residues in catalysis, and the reaction pH‐dependence is perturbed by each mutation. Solvent deuterium isotope effects indicate no rate‐limiting proton transfers. Crystal structures of D80N‐HsDNPH1 in unliganded and substrate‐bound states, and of unliganded D80A‐ and Y24F‐HsDNPH1 offer atomic level insights into substrate binding and catalysis. The results reveal a network of hydrogen bonds involving the substrate and the E104‐Y24‐D80 catalytic triad and are consistent with a proposed mechanism whereby D80 is important for substrate positioning, for helping modulate E104 nucleophilicity, and as the general acid in the first half‐reaction. Y24 positions E104 for catalysis and prevents a catalytically disruptive close contact between E104 and D80.

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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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Kinetics and Structure of a Cold-Adapted Hetero-Octameric ATP Phosphoribosyltransferase

Cited by 17 publications

References 49 publications

Catalytic and Anticatalytic Snapshots of a Short-Form ATP Phosphoribosyltransferase

Catalytic and Anticatalytic Snapshots of a Short-Form ATP Phosphoribosyltransferase

Human 2′‐Deoxynucleoside 5′‐Phosphate N‐Hydrolase 1: The Catalytic Roles of Tyr24 and Asp80

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

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