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

Fisher, Gemma; Thomson, Catherine M.; Stroek, Rozanne; Czekster, Clarissa Melo; Hirschi, Jennifer S.; Silva, Rafael G. da

doi:10.1021/acs.biochem.8b00559

Cited by 15 publications

(84 citation statements)

References 51 publications

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“…Pa ATPPRT catalysis follows an ordered mechanism in which PRPP binds first to the enzyme and involves a burst of on-enzyme PRATP formation in the first turnover followed by a lower steady-state rate dominated by product release. 11 When ATP and histidine are rapidly mixed with Pa ATPPRT preincubated with PRPP (Figure 2A), the amplitude of the burst phase, whose values are 3.49 ± 0.01 and 3.11 ± 0.01 μM in the presence and absence of histidine, respectively, is unchanged within experimental error. The subsequent steady-state rates, whose values are 16.36 ± 0.01 and 7.56 ± 0.01 μM s –1 in the absence and presence of histidine, respectively, reflect the inhibition.…”

Section: Resultsmentioning

confidence: 88%

Mapping the Structural Path for Allosteric Inhibition of a Short-Form ATP Phosphoribosyltransferase by Histidine

et al. 2019

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ATP phosphoribosyltransferase (ATPPRT) catalyzes the first step of histidine biosynthesis, being allosterically inhibited by the final product of the pathway. Allosteric inhibition of long-form ATPPRTs by histidine has been extensively studied, but inhibition of short-form ATPPRTs is poorly understood. Short-form ATPPRTs are hetero-octamers formed by four catalytic subunits (HisG S ) and four regulatory subunits (HisZ). HisG S alone is catalytically active and insensitive to histidine. HisZ enhances catalysis by HisG S in the absence of histidine but mediates allosteric inhibition in its presence. Here, steady-state and pre-steady-state kinetics establish that histidine is a noncompetitive inhibitor of short-form ATPPRT and that inhibition does not occur by dissociating HisG S from the hetero-octamer. The crystal structure of ATPPRT in complex with histidine and the substrate 5-phospho-α- d -ribosyl-1-pyrophosphate was determined, showing histidine bound solely to HisZ, with four histidine molecules per hetero-octamer. Histidine binding involves the repositioning of two HisZ loops. The histidine-binding loop moves closer to histidine to establish polar contacts. This leads to a hydrogen bond between its Tyr263 and His104 in the Asp101–Leu117 loop. The Asp101–Leu117 loop leads to the HisZ–HisG S interface, and in the absence of histidine, its motion prompts HisG S conformational changes responsible for catalytic activation. Following histidine binding, interaction with the histidine-binding loop may prevent the Asp101–Leu117 loop from efficiently sampling conformations conducive to catalytic activation. Tyr263Phe- Pa HisZ-containing Pa ATPPRT, however, is less susceptible though not insensitive to histidine inhibition, suggesting the Tyr263–His104 interaction may be relevant to yet not solely responsible for transmission of the allosteric signal.

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Section: Resultsmentioning

confidence: 88%

Mapping the Structural Path for Allosteric Inhibition of a Short-Form ATP Phosphoribosyltransferase by Histidine

et al. 2019

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“…Supporting a role in catalysis, Asp26 is highly conserved among TrmK orthologues; however, in B. subtilis TrmK, replacement of the equivalent residue Asp29 for alanine had a detrimental but not completely impairing effect on catalysis (9). Moreover, similar reactions where adenine N1 acts as a nucleophile have been proposed to proceed without general base catalysis (28), likely taking advantage of the transient negative charge on N1 due to a natural resonance structure of adenine (29).…”

Section: Discussionmentioning

confidence: 99%

Structure, dynamics, and inhibition of Staphylococcus aureus m¹A22-tRNA methyltransferase

Sweeney

Crowe

Kumar³

et al. 2021

Preprint

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The enzyme m1A22-tRNA methyltransferase (TrmK) catalyses the transfer of a methyl group from SAM to the N1 of adenine 22 in tRNAs. TrmK is essential for Staphylococcus aureus survival during infection, but has no homologue in mammals, making it a promising target for antibiotic development. Here we describe the structural and functional characterisation of S. aureus TrmK. Crystal structures are reported for S. aureus TrmK apoenzyme and in complexes with SAM and SAH. Isothermal titration calorimetry showed that SAM binds to the enzyme with favourable but modest enthalpic and entropic contributions, whereas SAH binding leads to an entropic penalty compensated by a large favourable enthalpic contribution. Molecular dynamics simulations point to specific motions of the C-terminal domain being altered by SAM binding, which might have implications for tRNA recruitment. Activity assays for S. aureus TrmK-catalysed methylation of WT and position 22 mutants of tRNALeu demonstrate that the enzyme requires an adenine at position 22 of the tRNA. Intriguingly, a small RNA hairpin of 18 nucleotides is methylated by TrmK depending on the position of the adenine. In-silico screening of compounds suggested plumbagin as a potential inhibitor of TrmK, which was confirmed by activity measurements. Furthermore, LC-MS indicated the protein was covalently modified by one equivalent of the inhibitor, and proteolytic digestion coupled with LC-MS identified Cys92, in the vicinity of the SAM-binding site, as the sole residue modified. These results these results identify a cryptic binding pocket of S. aureus TrmK and lay the foundation for future structure-based drug discovery.

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“…Here, we have shown that hinge twisting between domains I and II of the catalytic domain is the key in controlling catalytic activity and regulation. The catalytic domain of short-form ATP-PRT alone was shown to be poorly active (30,31,53). It is likely that HisZ activates and regulates short-form ATP-PRT, in an analogous fashion to the regulatory domain in long-form ATP-PRT, by controlling the hinge motions at the catalytic domain via noncovalent complex formation.…”

Section: Discussionmentioning

confidence: 99%

“…The ATP-PRT-catalyzed reaction between ATP and PRPP uses an ordered sequential mechanism, and therefore reaction occurs when both substrates are present in the active site. In long-form ATP-PRT, it has been shown that ATP binds first, followed by PRPP (28,29), whereas recent studies suggest that substrate binding may occur in the opposite order in short-form ATP-PRTs (30,31). After reaction, pyrophosphate is released, followed by phosphoribosyl-ATP (Fig.…”

Section: Introductionmentioning

confidence: 99%

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

Cited by 15 publications

References 51 publications

Mapping the Structural Path for Allosteric Inhibition of a Short-Form ATP Phosphoribosyltransferase by Histidine

Mapping the Structural Path for Allosteric Inhibition of a Short-Form ATP Phosphoribosyltransferase by Histidine

Structure, dynamics, and inhibition of Staphylococcus aureus m¹A22-tRNA methyltransferase

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

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

Cited by 15 publications

References 51 publications

Mapping the Structural Path for Allosteric Inhibition of a Short-Form ATP Phosphoribosyltransferase by Histidine

Mapping the Structural Path for Allosteric Inhibition of a Short-Form ATP Phosphoribosyltransferase by Histidine

Structure, dynamics, and inhibition of Staphylococcus aureus m1A22-tRNA methyltransferase

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

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Structure, dynamics, and inhibition of Staphylococcus aureus m¹A22-tRNA methyltransferase