Adenosine Tetraphosphoadenosine Drives a Continuous ATP

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Letter

Adenosine Tetraphosphoadenosine Drives a Continuous ATP-Release Assay for Aminoacyl-tRNA Synthetases and other Adenylate forming Enzymes Adrian J. Lloyd, Nicola J. Potter, Colin W. G. Fishwick, David I. Roper, and Christopher G Dowson ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/cb400248f • Publication Date (Web): 30 Jul 2013 Downloaded from http://pubs.acs.org on August 1, 2013

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ACS Chemical Biology

Adenosine Tetraphosphoadenosine Drives a Continuous ATPRelease Assay for Aminoacyl-tRNA Synthetases and other Adenylate forming Enzymes

Adrian J. Lloyd1*#, Nicola J. Potter2#, Colin W.G. Fishwick2, David I. Roper1 and Christopher G. Dowson1* 1

School of Life Sciences, University of Warwick, Gibbet Hill Road, Coventry, West Midlands,

CV4 7AL, UNITED KINGDOM and 2School of Chemistry, University of Leeds, Leeds, LS2 9JT, UNITED KINGDOM. *To whom correspondence should be addressed: Adrian J. Lloyd email: [email protected], Tel: +44 (0)2476 522568, Fax: +44(0)2476 523568 and Christopher G. Dowson email: [email protected], Tel: +44 (0)2476 523534, Fax: +44(0)2476 523568. #

Both authors contributed equally to the manuscript.

1

ACS Paragon Plus Environment


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Abstract Aminoacyl-tRNA synthetases are essential for the correct linkage of amino acids to cognate tRNAs to maintain the fidelity of protein synthesis.

Tractable, continuous assays are

valuable for characterizing the functions of synthetases and for their exploitation as drug targets. We have exploited the unexplored ability of these enzymes to consume adenosine tetraphosphoadenosine (diadenosine 5’,5’’’ P1 P4 tetraphosphate; Ap4A) and produce ATP to develop such an assay.

We have used this assay to probe the stereo-selectivity of

isoleucyl-tRNAIle and Valyl-tRNAVal synthetases, the impact of tRNA on editing by isoleucyltRNAIle synthetase (IleRS) and to identify analogues of intermediates of these enzymes that might allow targeting of multiple synthetases. We further report the utility of Ap4A based assays for identification of synthetase inhibitors with nM to mM affinities.

Finally, we

demonstrate the broad application of Ap4A utilization with a continuous Ap4A-driven RNA ligase assay. The ability of small molecules to distinguish between eukaryotic and prokaryotic protein synthesis has allowed development of many clinically valuable antibiotics(1) including mupirocin, which targets the active site of Gram positive IleRS(2). However, the lack of systemic efficacy of mupirocin, its inactivity against Gram negative and TB infections(2,3) and emergence of mupirocin resistance in MRSA and MSSA(2) has increased the need for new synthetase inhibitors. Many aminoacyl-tRNA synthetases (AaRSs) cannot bind the correct amino acid accurately enough to ensure the fidelity of protein synthesis(4). Therefore these enzymes remove incorrectly aminoacylated intermediates and tRNAs with pre and post-transfer editing mechanisms. These are partitioned between the active site (pre-transfer) and a posttransfer editing domain(4).

Antimicrobial targeting of the post-transfer editing domain of

leucyl-tRNALeu synthetase (LeuRS) from Gram negative pathogens has been abandoned because of the development of resistance in clinical trials due to mutation(5). This result was 2


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heralded by the impact of mutation of the post-transfer editing of Escherichia coli LeuRS which unveiled a pre-transfer-editing capability(6). All AaRSs activate their cognate amino acids with ATP to form an aminoacyladenylate (Figure 1a). AaRSs then catalyse the attack of this intermediate by tRNA to form the correct aminoacyl-tRNA product(4). However, in the absence of tRNA, AaRSs can cleave this aminoacyl-adenylate with pyrophosphate. monitored as [32P]-pyrophosphate Alternatively,

Consequently, AaRS activity can be

exchange into ATP. (Figure 1a reaction 1)(7).

aminoacyl-tRNA formation (Figure 1a, reaction 2) can be followed with

radiolabelled amino acid by trichloroacetic acid precipitation of the radiolabelled aminoacylated tRNA product(7). However, utilization of radiolabels prevents continuous monitoring of AaRS activity and is expensive, which often precludes high throughput and saturating substrate experiments(7). This and the success of mupirocin suggest significant advances in inhibitor screening and AaRS enzymology could be leveraged by continuous non-radioactive assays that monitored AaRS aminoacyl adenylate turnover. We initially identified the assay in(8) as a starting point.

Here, the pyrophosphate

attacking the aminoacyl adenylate intermediate exchanged for the imidodiphosphate moiety of 5'-adenylyl-β,γ-imidodiphosphate (ADPNP) in the presence of the amino acid to generate ATP that could be assayed spectrophotometrically (Supporting Information Figure 1 (Figure S1a)). We therefore obtained an assay for E. coli and Streptococcus pneumoniae alanyltRNAAla and S. pneumoniae seryl-tRNASer synthetases (AlaRS and SerRS) that we characterised (Table S1 and Figure S2a-i). However the E. coli class I IleRS and valyl-tRNAVal synthetases (ValRS) could not utilise ADPNP (Figure S1b), possibly because of differential modes of ATP binding to class I and II synthetases(9). Furthermore, the low micromolar ADPNP KmApp of E. coli AlaRS (Table S1) reduced the sensitivity of the assay to competitive inhibitors. To enable access to the 3



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enzymology of both class I and II AaRSs, that was highly sensitive to hit detection, we sought another adenylate to support pyrophosphate exchange activity. The synthetases discussed above synthesise Ap4A where the aminoacyl adenylate formed with the concomitant generation of pyrophosphate, is cleaved by a second ATP(12) to form Ap4A (Figure 1a, reaction 3)(10).

Reversal of this reaction would generate two

molecules of ATP which could be continuously detected by spectrophotometry, fluorometry or bioluminescence (Figure 1b). Assays following conversion of Ap4A to ATP require detection of ATP in the presence of Ap4A.

We found that glucose phosphorylation with 0.1 mM ATP by hexokinase when

coupled to glucose 6-phosphate dehydrogenase to generate NADPH (Figure 1b) caused an instantaneous increase in absorbance at 340 nm in the presence of 6.33 mM Ap4A which itself, caused no significant absorbance change. The absorbance increase was consistent with the ATP concentration added to the assay.

This suggested the coupling system

discriminated between ATP and Ap4A and was not significantly inhibited by the dinucleotide. We then tested the ability of E. coli AlaRS, E. coli ValRS and E. coli IleRS to generate ATP from Ap4A. All three enzymes showed linear Ap4A, pyrophosphate, synthetase and amino acid dependent ATP production as reported at 340nm (Figure 2a-c). The linear initial rates in Figure 2a-c indicated the products of the coupling enzymes (Figure 1b) did not significantly impact on AaRS activity. To prove that the assay followed the pyrophosphorolytic cleavage of Ap4A we followed

Ap4A consumption by S.pneumoniae AlaRS in the presence of L-alanine and

excess pyrophosphate. At the end of the reaction, two ATP were produced per Ap4A (Figure 2d), consistent with the stoichiometry of this reaction. Amino acid-dependent AaRS catalysed Ap4A pyrophosphorolysis

would not be

limited by the amino acid which is regenerated per catalytic cycle (Figure 1b). Therefore, we 4


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assayed IleRS with limiting (10 µM) L-isoleucine. The absorbance of the assay extended beyond that where L-isoleucine was consumed (0.062 AU), to that consistent with consumption of the next limiting substrate in the assay (pyrophosphate; Figure 2c) and regeneration of L-isoleucine. Having characterised AaRS pyrophosphorolysis of Ap4A, we examined the kinetics of this process for IleRS, ValRS and AlaRS. Dependences of activity on Ap4A, cognate amino acids and pyrophosphate were hyperbolic (Figures 2e and S3a-i). In many of these assays, the AaRS concentration approached that of the substrate (especially pyrophosphate). Therefore, we fitted the data to a rate equation that took this into account (Table 1 legend). This analysis revealed that for E. coli AlaRS, the KmApp for Ap4A was 204-fold greater than that for ADPNP (Tables 1 and S1, Figures S2h and S3h) . Comparison of Table 1 and Figure 3 with published data suggested that the KmApp of E. coli AlaRS, IleRS and ValRS for Ap4A was 16.8-(11), 45.2-(12) and 21.6-fold(13) greater than for ATP.

Thus, Ap4A utilization

afforded a continuous assay, with kinetic constants that would be more sensitive to inhibitor detection than assays that utilised ATP. The KmApp values in the Ap4A assay for the cognate amino acids for ValRS, AlaRS and IleRS (Table 1, Figures 2e, S3f, i and c) were elevated by 101-fold, 70.2-fold and 9.03fold respectively relative to published data(11,14,15). This may have resulted from a reduced affinity of the amino acid for synthetases bound to Ap4A, relative to that in the presence of ATP. As also deduced from the Ap4A kinetics, the sensitivity of assays to amino acid or aminoacyl-adenylate synthetase-targeted inhibitors would be expected to be vastly enhanced by the relatively high KmApp values of these enzymes for their cognate amino acids. ValRS(16), IleRS(16) and AlaRS(17) form adenylates and aminoacyl-tRNA species with non-cognate amino acids. We therefore tested the ability of these enzymes to utilise Ap4A with L-threonine, L-valine and L-serine, the respective non-cognate editing substrates of these enzymes(16,17). Activation of L-valine by IleRS and L-serine by S. pneumoniae AlaRS 5



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was easily detectable (Figure 2f and Figure S4). The efficiencies ( kcatApp/KmApp values) of Lvaline and L-isoleucine activation by E. coli IleRS (Table 1) were 1:207.5, in excellent agreement with previous data (1:200)(18) and consistent with the requirement for editing by IleRS.

However, Ap4A consumption by ValRS was barely detectable with 15 mM L-

threonine. It is probable that the reduced affinity for both non-cognate amino acid and the substitution of ATP by Ap4A severely reduced ValRS activity. tRNAIle stimulates IleRS editing(16) (Figure S5a). Therefore, we assessed the ability of Ap4A to support this process. tRNA stimulated Ap4A utilization by IleRS with L-valine but not L-isoleucine (Figure S5b). (Supporting Information section 4.1 (SI 4.1)) This showed that Ap4A assays could provide a valuable non-radioactive alternative to the current radiolabel and TLC-based procedures used to study this process(16). We extended our IleRS and ValRS studies to the utilization of D-isoleucine and Dvaline in the Ap4A assay.

D-valine did not support ValRS or IleRS activity, as previously

observed(19,20). In contrast, D-isoleucine was activated by IleRS (Figure 2(g)) where IleRS was only 189.2-fold more efficient with the L-stereoisomer (Table 1).

This level of

discrimination is similar to that between L-isoleucine and L-valine which has required the evolution of editing mechanisms to eliminate mis-valylation of tRNAIle

(4,16,18)

.

How mis-

activation of D-isoleucine by IleRS is dealt with is relevant because D-isoleucine is found in the peptidoglycan of stationary phase bacterial cultures(21). This raises the possibility that Disoleucyl-tRNAIle may challenge the stereochemical fidelity of protein synthesis, a possibility enhanced by our inability to demonstrate any editing activity with respect to D-isoleucine by E. coli IleRS (Lloyd unpublished data). To validate the Ap4A assay for inhibitor detection, we tested the response of AlaRS, ValRS, SerRS and ThrRS to a series of aminoacyl-adenylate analogues. Data (Figure S6) were analysed as in(22) to determine Ki values for inhibition (Equation 2 and 3, Figure S7 Legend), as AaRS and inhibitor concentrations were comparable in some of the assays. 6


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Mupirocin is a tight binding analogue of isoleucyl-adenylate(2). In the Ap4A assay, mupirocin was a potent IleRS inhibitor (Figure 2c) with an IC50 of 0.35 ± 0.06 µM (Figure S6a,e) approximating to half the concentration of IleRS (1.13 µM), where the Ki for this interaction was 0.52 ± 0.12 nM (Figure S7a). Conversely, mupirocin was a weak ValRS inhibitor (37% at 0.5 mM), consistent with the specificity of the drug towards prokaryotic IleRS. Aminoacyl-5’-sulphamoyl adenosines are potent AaRS inhibitors(23) and E. coli AlaRS was inhibited by alanyl-5’-sulphamoyl adenosine (Figure 2a) with an IC50 of 0.403 ± 0.005 µM (Figure S6b,e, Figure S7b).

The impact of cognate adenylate analogues on their

cognate AaRSs as shown here with AlaRS and IleRS confirmed the utility of the Ap4A assay. We then probed the ability of aminoacyl-sulphamoyl adenosines to inhibit both cognate and non-cognate AaRSs. Our data strongly suggest that multiple targeting of AaRSs by single potent adenylate based inhibitors is achievable (SI 4.2). The Ap4A assay was originally developed to assess inhibition by molecules selected or designed by in silico drug design. We used the de novo molecular design programme SPROUT(24) to identify small molecules pockets of E. coli IleRS and ValRS.

that bound to the aminoacyl-adenylate binding Focussed libraries of molecules were synthesised

based around these scaffolds (SI 1.1,1.2,2.1,2.2). This and additional virtual high throughput screening (vHTS) generated a library of 63 compounds (SI 1.3, Tables S2-S5, Figure S9). Screening assays sensitive to ValRS and IleRS inhibitors with as low as millimolar affinities were designed (SI 4.3) and implemented to detect inhibitors of these enzymes within this library. Although we were unable to identify dual targeted inhibitors by this route, we identified

two

IleRS

inhibitors

we

synthesised

(SI

2.2):

N-tert-butyl-2-(3-{[3-(tert-

butylcarbamoyl-methyl)benzyl-amino]-methyl}-phenyl)-acetamide (1) and N-isopropyl-2-(3{[3-(isopropyl-carbamoyl-methyl)-benzylamino]-methyl}-phenyl)-acetamide (2) (Table S6) with Z’-factors(25) of 0.73 and 0.55 and IC50 values of 0.22 ± 0.035 mM and 0.895 ±0.057 7



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mM respectively (Figure 3a and b). These molecules exerted no effect on the hexokinase/6phospho-gluconate dehydrogenase coupling system. Both 1 and 2 were competitive inhibitors against L-isoleucine and Ap4A (Figures 3c-f), suggesting that they bound to IleRS at the site they were designed to target. The Ki values of 1 and 2 (Figures 3c-f) suggested they bound weakly to IleRS, but are consistent with the starting point for fragment based drug design(26). Screening of this library with ValRS (Table S7) identified 3 (4-Amino-2-[2-oxo-2-(3phenyl-isoxazol-5-yl)ethylsulphanyl]-pyrimidine-5-carboxylic acid)) as a ValRS inhibitor (Z’ factor = 0.74) that was not an IleRS inhibitor. 3 did not inhibit the coupling enzymes at 1 mM and on re-screening displayed a sigmoid dependence of inhibition on concentration (IC50 = 1.51 ± 0.32 mM, Hill coefficient of 3.65 ± 0.68; Figure S10d)). To gain additional insight into molecules that might inhibit ValRS, we identified three triazinyl dyes: Procion Red HE-3B, Procion Green HE4BD (Table S7) and Trypan Blue that inhibited the enzyme. The effect was specific (the coupling enzymes were unaffected by up to 0.1 mM dye as judged by the response of an inhibited ValRS assay to 0.1 mM ATP). Furthermore, these dyes did not inhibit IleRS. IC50//Ki values(22) for Procion Red HE-3B, Procion Green HE4BD and Trypan Blue were 2.14 ± 0.29 µM/0.49 ± 0.06 µM, 6.98 ± 1.23 µM/2.21 ± 0.52 µM and 19.2 ± 8.6 µM/13.2 ± 1.47 µM respectively (Figures S10a-c). Procion Green HE4BD inhibits methionyl-tRNAMet synthetase(27) suggesting that this dye could facilitate development of multi-targeted AaRS inhibitors.

Trypan blue has anti-

trypanosomal activity(28). Our data suggest that this molecule could target ValRS, which itself may be a viable strategy in treatment of trypanosomiasis. The utility of the Ap4A assay led us to investigate its use with other adenylate utilizing enzymes.

T4 RNA ligase type 1 self adenylates a lysine residue (K99) prior to RNA

ligation(29) and also synthesises Ap4A(30). Therefore we tested the ability of RNA ligase to 8


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generate ATP from Ap4A as we would anticipate the enzyme to self-adenylate using Ap4A to generate ATP.

Subsequently, pyrophosphate could cleave the enzyme lysyl adenylate

generating a second ATP (Figure S11a). As anticipated, T4 RNA ligase catalysed the pyrophosphorolysis of Ap4A in a manner dependent on enzyme, pyrophosphate and Ap4A ( Figure S11b), which showed a linear rate that is stable for at least 30 minutes. This demonstrates the potentially great utility of Ap4A as an assay with respect to other adenylate forming enzymes and may be an improvement on current ligase adenylation assays (SI 4.4 and SI. 4.6). Other continuous AaRS assays have been developed, including the method(8) which initiated this work. These assays are compared to the Ap4A assay in the SI 4.5 and SI. 4.7. Finally, many drug targets turnover Ap4A(31). Indeed in TB there are over sixty enzymes that utilise adenylate intermediates(32) Furthermore, with respect to Ap4A assay implementation, NADPH utilization as a reporter of activity suggests an additional fluorescence format for these assays, and generation of ATP from Ap4A indicates a potential

luciferase based

luminescence readout, yielding three separate techniques for Ap4A utilization assays. Methods Methods are incorporated into Supporting Information (SI 3) Associated Content Methods, additional figures and tables as described in the text are available free of charge in Supporting Information via the Internet at http://pubs.acs.org. Acknowledgments We thank D. Söll for critical reading of the manuscript and the MRC for financial support. A. Lloyd thanks the Birmingham-Warwick Science City Translational Medicine Initiative for support. 9



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Fraga, H., and Fontes, R. (2011) Enzymatic synthesis of mono and dinucleoside polyphosphates. Biochim. Biophys. Acta. 1810, 1195-1204.

32. Duckworth, B. P., Nelson, K. M., and Aldrich C. C. (2012) Adenylating enzymes in Mycobacterium tuberculosis as drug targets Curr. Top. Med. Chem. 12, 766–796. 33. Segel, I. H. (1975) Enzyme Kinetics: Behaviour and Analysis of Rapid Equilibrium and Steady-state Enzyme Systems. pp. 18–99, Wiley-Interscience, New York.

Table Legends Table 1: Characterization of the substrate dependence of E. coli AlaRS, IleRS and ValRS on the rate of pyrophosphorolysis of Ap4A

Assays were performed as

noted in the table. Due to the high concentrations of enzyme used relative to the lower concentrations of some substrates (particularly pyrophosphate) all data for consistency were fitted to an equation which takes the concentration of enzyme in the assay into account(33): [E] and [S] are enzyme and substrate concentrations: Vo =

kcatApp 2

([E] + [S] + KmApp)2 – (4.[E].[S])

13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 20

vo vs [S] plots (Figures 2(e)-(g), S2 and S3) were assessed for goodness of fit using the correlation coefficient (r2) which in all cases was >0.92. Figure Legends Figure 1: Mechanism of Ap4A production and exploitation of its consumption by AaRSs.

Figure 1a: Catalytic activities of AaRSs. Formation of the central

aminoacyl-adenylate intermediate and inorganic pyrophosphate from ATP and an amino acid(4), and reaction of this intermediate with the C-2 or C-3 hydroxyl of the 3’ ribose of tRNA (reaction 2) to form an aminoacyl-tRNA. The aminoacyl-adenylate can also be cleaved by pyrophosphate (reaction 1), displacing the amino acid to regenerate ATP, or by the oxygen atom appended to the γ-phosphorus of a second molecule of ATP which results in the amino acid-dependent formation of adenosine tetraphospho-adenosine (Ap4A; reaction 3).

Figure 1b:

Reversal of Ap4A

synthesis by AaRS. Cleavage of Ap4A by an amino acid substrate and pyrophosphate yielding two molecules of ATP which can be continuously monitored by coupling to NADP+ reduction to NADPH. Figure 2:

Kinetics, stoichiometry and amino acid specificity of AaRS-catalysed

pyrophosphorolysis of Ap4A to ATP.

Panel (a) – (d); Time courses of AaRS

pyrophosphorolysis PPi denotes pyrophosphate. Background rates were followed before addition of the missing component of the assay at arrow. Panel (a): E. coli AlaRS in the presence of 0.41 mM Ap4A, 2 mM L-alanine, 20 µM pyrophosphate and 0.92 µM AlaRS; Panel (b) E. coli ValRS in the presence of 0.388 mM Ap4A, 4 mM L-valine, 20 µM pyrophosphate and 2.41 µM ValRS; Panel (c) E. coli IleRS in the presence of 0.7 mM Ap4A, 10 µM L-isoleucine, 20 µM pyrophosphate and 2.4 µM ValRS. For panels (a) - (c) assays were run without (maroon trace, DMSO control) or with 5 µM alanyl-5’-sulphamoyl adenosine (ASA), 50 µM threonyl-5’-sulphamoyl adenosine (TSA) or 5 µM mupirocin (blue trace). Panel (d): Stoichiometry of Ap4A 14


Page 15 of 20

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conversion to ATP in the presence of L-alanine and S. pneumoniae AlaRS. Reaction was allowed to approach equilibrium in the presence of 1.8 µM AlaRS, 0.471 mM pyrophosphate and 27.2 µM Ap4A. Final [ATP] inferred from the ∆A340 on completion of the reaction was 59.2 µM, implying conversion of Ap4A to 2.16 equivalents of ATP. Panel (e) – (g): dependence of initial velocity of IleRS on [L-Isoleucine], [L-valine] and [D-Isoleucine] respectively. For panels (e) to (g) Assays were performed at [cosubstrate] and [enzyme] in Table 1. Data were fitted to the equation in the figure legend to Table 1(22) Figure 3: Evaluation of 1 and 2, IleRS inhibitors identified with the Ap4A assay. [IleRS] = 3.14 µM IleRS, [pyrophosphate] = 20 µM. Panels (a) and (b) dependence of inhibition on 1 or 2 respectively. [Ap4A] and [L-isoleucine] were 0.7 mM and 10 µM respectively. Panels (c) and (d): (IleRS activity)-1 vs. [L-isoleucine]-1 in the presence of 0-3 mM 1 (c) or 0-3.6 mM 2 (d). Panels (e) and (f) ): (IleRS activity)-1 vs. [Ap4A]-1 in the presence of the same range of 1 (e) or 2 (f) concentrations. r2 values for data collected at each [inhibitor] are in purple. Inserts in panels (c) to (f) are secondary plots of slopes of reciprocal plots vs. [inhibitor]. Ki values were determined from the abscissa intercept of secondary plots.

15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Page 16 of 20

Table 1

AaRS

AlaRS

IleRS

ValRS

[AaRS]Assay

Nonvaried substrate

[Nonvaried substrate] (mM)

Varied Substrate

KmApp (µ µM)

kcatApp (min-1)

kcatApp/KmApp (min-1.µ µM-1)

Goodness of fit (r2)

0.92 µM

L-Alanine

10.0

L-Alanine

14200 ± 1540

39.3 ± 1.93

0.00280

0.984

0.92 µM

Ap4A

3.99

Ap4A

870 ± 171

21.8 ± 1.41

0.0251

0.961

0.92 µM

PPi

0.471

PPi

12.2 ± 1.62

19.5 ± 0.800

1.600

0.979

1.13 µM

L-Isoleucine

14.4

L-Isoleucine

36.1 ± 3.12

6.960 ± 0.180

0.193

0.994

1.13 µM

Ap4A

5.00

Ap4A

6330 ± 929

16.50 ± 1.29

0.00261

0.990

1.13 µM

PPi

0.470

PPi

2.46 ± 0.470

7.53 ± 0.360

3.060

0.923

0.74 µM

L-Valine

3950 ± 694

3.62 ± 0.210

0.000930

0.980

0.74 µM

D-Isoleucine

4090 ± 411

4.17 ± 0.174

0.00102

0.993

1.10 µM

L-Valine

14.4

L-Valine

12700 ± 1700

10.70 ± 0.790

0.000840

0.991

1.10 µM

Ap4A

1.53

Ap4A

1740 ± 161

9.92 ± 0.377

0.00570

0.978

1.10 µM

PPi

0.471

PPi

1.80 ± 0.190

4.08 ± 0.0870

2.267

0.988

16


Page 17 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1a O NH2

NH2

Adenosine tetraphospho Adenosine (Ap (AP44A) N

N

O OH OH

O O

O O O P P P O OO OO O

OH OH

N

N

O O O O O O P P P P O OO O O O O

Amino Acid

R

N

N NH2

N

N

N

N

N

NH2

O

Pyrophosphate

O O P OO O P O

ATP

N

O OH OH

H2N

R

O

NH2

O O

3

NH2

O

N

O O

N

N

O P OO O

O O

O

O

P P P O OO OO O

O OO O P P O O

O

Central Aminoacyl Adenylate Intermediate

OH OH

2 tRNA

R

NH2

Aminoacyl-tRNA

H2N O

N

N

N

N

O O

5’-AMP

O O O P P P O OO OO O

OH OH

O

O

O

NH2

OH OH N

N

ATP*

N

N

R

N

N

N

1

N

N

R

NH2

O

NH2

O P OO

OH OH

Figure 1b Pyrophosphate -O

Amino acid O NH2

-O

N

N

NH3+

N O OH OH

N

N

R

N

OH OH N

O OP P -O O O O-

NH2

O PO O -O O O OP P P O -O O O O -O

Ap AP4A 4A:

O

-O R

N NH2

R

OH OH O -O O O OP -O P O P O O -O

NH2

NH3+

N N

N

N

ATP

N

2 Glucose

2 6-Phosphogluconate

Hexokinase

2 Glucose-6-Phosphate NH2

ADP

2 NADPH + 2 H

+

N

+

2 NADP

HO

O

N

O

P P -O O O O-

∆A340

N

O OH OH

17


N N

2

O O O -O P P P -O O O O- O

O O

OH OH

NH2

Glucose 6-phosphate Dehydrogenase

NH3 +

N

N

AaRS

O

ATP

O

O

OH OH

N

N

Amino acid

O

P -O O

O

O N

N


Figure 2

0.24 0.20

0.24

(a) E. coli AlaRS

0.08

20 µM PPi

20 µM PPi

0.12 0.08

0.04 4

8

12

16

+ 50 µM TSA

0.04

+ 5 µM ASA

0.00 0

0.00 0

20

2

4

Time (min) 0.20

(c) E. coli IleRS

DMSO

20 µM PPi

0.08

Net: AP4A + PPi

30

0 8

12

14

12

16

20

0

Time (min)

20

40

(e)

59.2 µM ATP

2 ATP

60

80

100

Time (min)

25 mM L-Alanine

(f)

6

Alanine

Initial [Ap4A] = 27.2 µM Final [ATP] = 59.2 µM Expected [ATP]/[AP4A] = 2.00 Observed [ATP]/[AP4A] = 2.18

15

+ 5 µM mupirocin

4

10

45

0.04 0.00 0

8

(d) S. pneumoniae AlaRS

60

µ M ATP

0.12

6

Time (min)

0.16

∆ A340

DMSO

0.16

∆ A340

∆ A340

0.16 0.12

(b) E. coli ValRS

0.20

DMSO

(g)

3.2

3.2

2.4

2.4

4 3 2

v0 (min-1)

vo (min-1)

5

Vo (min-1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 20

1.6 0.8

1.6 0.8

1 0 0.00

0.04

0.08

0.12

0.16

[L-Isoleucine] (mM)

0.20

0.0 0

3

6

9

12

15

[L-Valine] (mM)

18


18

0.0 0

3

6

9

[D-Isoleucine] (mM)

12

Page 19 of 20

Figure 3

(a)

(b) 75

100 % Inhibition

% Inhibition

60 2

75

(r = 0.971) O

50

(IC50 = 0.220 ± 0.035 mM)

N H

O

H N

25

(r2 = 0.923)

45

0 0.0

0.4

0.8 1.2 [1] (mM)

1.6

Slope (Km app/Vmaxapp) µ M.min) (µ

250

Ki = 0.184 mM

200

r2 = 0.985

(d) [1] (mM)

50 0 0.0 0.6 1.2 1.8 2.4 3.0 [1] (mM)

1.0 1.5 [2] (mM)

3.5 2.0

2.5

Ki = 0.351 mM

[2] (mM)

r2

5 4

v-1 (min)

2.25 1.80 1.50 1.00 0.75

2.0

3.6 0.997

r2

3.00 0.997

5.0

v (min)

0.5

r2 = 0.971 0.934

100

-1

N H

Inhibition with respect to isoleucine

150

6.5

O

H N

0 0.0

2.0

(IC50 = 0.895 ± 0.057 mM)

N H

15

Inhibition with respect to isoleucine

(c)

O

30

N H

0.995 0.999 0.987 0.995 0.997

3.0 0.993

[2] (mM)

2.4 0.986

3

1.8 0.997

2

1.0 0.975 0

1

0.966

0.00 0.866

0.5 -0.01

-1.0

0.01

0.000 0.025 0.050 0.075 0.100 0.125

0.02

-1 [L-Isoleucine] µM-1) [Isoleucine] (µ µM-1) (µ

-1 (µ µM-1) -1

[L-Isoleucine] [Isoleucine] (µ µM )

12

9.0

9

Inhibition with respect to Ap4A

(f)

Ki = 0.140 mM r2 = 0.959 [1] (mM)

3 0 0.0 0.6 1.2 1.8 2.4 3.0 [1] (mM)

r2

9.0 7.5

3.00 0.984

7.5

[2] (mM)

2.25 0.994

6.0

-0.2

1.80 0.989 1.50 0.982

4.5 3.0

0.75 0.986 0.30 0.999 0.00 0.902

1.5 -1.5 -3.0

0.2

Ki = 0.730 mM r2 = 0.930 0.934

6

v-1 (min)

(e)

Slope (Km app/Vmaxapp) (mM.min)

Inhibition with respect to Ap4A

v-1 (min)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.4

[Ap [Ap4A] 4A]

-0.5

-1

-1-1 (mM (mM ))

r2

3.6

0.988

3.0

0.978

2.4 1.8

0.992 0.993

1.0

0.994

0

0.998

6.0 4.5 3.0 1.5

0.6

[2] (mM)

0.0

0.5

1.0 -1 -1

1.5

2.0 -1 [Ap

[Ap4A] [NJP09] (mM ) 4A]

19


2.5

3.0

(mM-1)


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Page 20 of 20

Adenosine Tetraphosphoadenosine Drives a Continuous ATP

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