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The dual activity of Quinolinate Synthase: Triose Phosphate Isomerase and dehydration activities play together to form quinolinate Debora Reichmann, Yohann Couté, and Sandrine Ollagnier de Choudens Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.5b00991 • Publication Date (Web): 12 Oct 2015 Downloaded from http://pubs.acs.org on October 13, 2015
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Biochemistry
The dual activity of Quinolinate Synthase: Triose Phosphate Isomerase and dehydration activities play together to form quinolinate Debora Reichmann1,2,3, Yohann Couté4,5,6, Sandrine Ollagnier de Choudens1,2,3,* 1 Univ. Grenoble Alpes, iRTSV-LCBM, F-38000 Grenoble, France; 2 CNRS, iRTSV-LCBM, F-38000 Grenoble, France; 3 CEA, iRTSVLCBM-Biocat, F-38000 Grenoble, France; 4 Université Grenoble Alpes, iRTSV-BGE, Grenoble 38000, France ; 5 CEA, iRTSV-BGE, Grenoble 38000, France ; 6 INSERM, BGE, Grenoble 38000, France.
KEYWORDS NAD, Quinolinate, triose phosphate isomerase, NadA, mechanism Supporting Information Placeholder ABSTRACT: Quinolinate synthase (NadA) is an Fe4S4 clustercontaining dehydrating enzyme involved in the synthesis of quinolinic acid (QA), the universal precursor of the essential coenzyme nicotinamide adenine dinucleotide (NAD). The reaction catalyzed by NadA is not well understood and two mechanisms have been proposed in the literature differing in the nature of the molecule (DHAP or G-3P) that condenses with iminoaspartate (IA) to form QA. In this article, using biochemical approaches, we demonstrate that DHAP is the triose that condenses with IA to form QA. The NadA capacity to use G-3P is due to its previously unknown triose phosphate isomerase activity.
Nicotinamide adenine dinucleotide (NAD) is an essential and ubiquitous cofactor involved in a myriad of redox and non-redox reactions1-3. The initial steps in the biosynthesis of NAD vary considerably between prokaryotes and eukaryotes, although both pathways involve the common intermediate quinolinic acid (QA). In most eukaryotic organisms, QA is produced by the oxidative degradation of L-tryptophan by a 5-enzymes pathway4. In most prokaryotes, including Escherichia coli, and in plants, QA is generated by a unique condensation reaction between dihydroxyacetone phosphate (DHAP) and iminoaspartate (IA). This reaction requires the concerted actions of two proteins, L-aspartate oxidase (NadB) that first converts L-aspartate to iminoaspartate and quinolinate synthase (NadA) which condenses IA with DHAP to form QA. Besides these de novo pathways for NAD synthesis, a salvage pathway exists in some organisms allowing NAD to be recycled from nicotinic acid (NA) and nicotinamide (Nm)5. However, some pathogens such as Mycobacterium leprae and Helicobacter pylori have been reported to lack this salvage pathway6. The different pathways for QA biosynthesis in most prokaryotes and eukaryotes, combined with the fact that the salvage pathway is absent in some pathogenic microorganisms makes NadA an ideal target in the search for specific antibacterial drugs. As-isolated NadA protein binds one oxygen-sensitive Fe4S42+ cluster that is essential for its activity7,8. The cluster is bound by only three strictly conserved cysteines9, reminiscent of dehydratases such as aconitase, “Radical-SAM” enzymes or IspG/H enzymes, where the fourth coordination site is accessible and occupied by the cognate (co)substrate10-12. Using a structural analogue of QA, we demonstrated that the NadA Fe/S cluster also contains an accessible iron site which is essential for its activity,
strongly suggesting that the chemistry of QA formation occurring at the Fe4S42+ cluster takes place on this iron site13. Recently, the crystal structure of the active Fe4S4-bound NadA from Thermotoga maritima was solved at 1.65 Å resolution14. This structure confirmed the presence of an accessible iron site, coordinated by a water molecule14 and has allowed the identification of key aminoacids in the active site. The structure revealed a long tunnel connecting the molecular surface of the enzyme to the H2Ocoordinated iron of the cluster where substrates could be accommodated.
Scheme 1: Mechanisms proposed for quinolinic acid biosynthesis from DHAP or G-3P and iminoaspartate as published by Sakubara et al. (A)15 and by T. Begley (B)5. The iron atom from the Fe4S4 that functions as a Lewis acid in the reaction is shown (Fe). The reaction catalyzed by NadA is not yet known in terms of intermediates, but two mechanisms have been proposed for the synthesis of QA from DHAP and IA5,15. These mechanisms are summarized in Scheme 1 where the role of the iron atom from the Fe4S4 cluster which is available to act as catalyst is also shown. Both mechanisms involve the removal of 2 mol of water and 1 mol of inorganic phosphate16, but they differ in how the two substrates are condensed. In mechanism (A), based on early labeling studies17, condensation occurs between C1 of DHAP and C3 of iminoaspartate, leading to early release of Pi. In mechanism (B), in contrast, DHAP is first isomerized into glyceraldehyde 3phosphate (G-3P) which then condenses through its C1 with the amino group of iminoaspartate, resulting in later release of the phosphate group. So far, no experimental support has been provided for either of these mechanisms5,15. The involvement of G3P as a substrate was initially suggested based on experiments using partially purified NadA and NadB enzymes and labeled substrates18,19.
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DHAP G-3P
kcat (min-1) 13.9 9.9
KM (mM) 0.74 ± 0.08 0.64 ± 0.12
keff (M-1. s-1) 313 258
Table 1. Kinetic parameters determined for DHAP and G-3P. E. coli NadA (10 µM) was incubated for 20 min at 37°C with 10 mM L-aspartate, 25 mM fumarate, 10 µM NadB, and DHAP or G-3P at concentrations ranging from 0 to 8 mM in 0.1 M Hepes pH 7.5, 0.05 M KCl under anaerobic conditions (n≥4). G-3P and DHAP are triose phosphate molecules that can interconvert within cells thanks to the TPI activity of triose phosphate isomerase enzymes. TPI are extremely efficient cellular enzymes which all favor DHAP formation. TPI from eukaryotes have a kcat of 2.5-5.1 x 105 min-1 from G-3P to DHAP and a kcat of 2.5-5.2 x 104 min-1 for the reverse reaction20,21. The TPI from Escherichia coli, TpiA, has a similar activity (kcat = 6.1 x 103 min1 from G-3P to DHAP)22. We investigated whether NadA is endowed with TPI activity based on spectrophotometric assays involving NAD/NADH-dependent reactions. For conversion of G-3P to DHAP, DHAP production was measured by tracking the consumption of NADH by glycerol-3-phosphate dehydrogenase (GPDH) (Fig. S1 in Supporting Information (SI)). The reverse reaction was monitored using NAD+-dependent glyceraldehyde-3phosphate dehydrogenase (GAPDH)23 (Fig. S1 in SI). We measured the TPI activity of E. coli NadA under aerobic conditions against a positive control, commercial rabbit muscle TPI. As shown in Figure 1, NadA can convert G-3P into DHAP and back again. Like all TPI enzymes, it is more efficient in the direction of DHAP production (1.2±0.05 µmol/min/mg against 0.084±0.02 µmol/min/mg for G-3P production). Therefore, NadA exhibits TPI activity in vitro, although this activity is much less potent than that of the rabbit muscle TPI (10 000-fold lower) (Figure 1). Interconversion between DHAP and G-3P is enzymedependent since with the reaction time we used, practically no DHAP or G-3P production could be detected using G-3P and DHAP as sole components of the reaction mixture, respectively. Indeed, with G-3P 4.6% conversion was measured, while incubation of DHAP resulted in 0.6% conversion after 30 min. (Fig. S2 in SI). The TPI activity measured with E. coli NadA was specific, as E. coli ferredoxin, an Fe2S2 protein, and commercial
E. coli BSA displayed no activity at all (data not shown). Since the Fe4S4 cluster of NadA converts to an Fe2S2 cluster under aerobic conditions, the isomerization experiment was repeated anaerobically. No significant change in activity was observed (0.31±0.002 (aerobiosis) vs 0.33±0.01 (anaerobiosis) µmol/min/mg for DHAP production and 0.028±0.003 (aerobiosis) vs 0.022±0.006 (anaerobiosis) µmol/min/mg for G-3P production), suggesting that the Fe4S4 cluster is not important for the TPI activity of NadA (Fig. S3A in SI). 10000
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To gain insight into NadA reaction mechanism, in particular to determine which mechanism (A or B) is more likely, we compared G-3P vs DHAP as substrate in the presence of IA for QA formation using purified proteins. Through biochemical methods we demonstrated that purified NadA from different organisms has a triose phosphate isomerase (TPI) activity catalyzing the reversible isomerization of G-3P into DHAP in an Fe/S dependent manner. However, only DHAP can condense with IA to produce QA. Thus, our results suggest that mechanism A is used for QA formation. Physiological explanations for this dual activity are discussed. First, we measured quinolinate synthase activity in vitro by mixing purified NadA, NadB and L-aspartate (precursor of IA) with either DHAP or G-3P. A kcat of 9.9 min-1 (Table 1) was determined in the presence of G-3P and IA, and a similar kcat (13.9 min-1) was determined with DHAP and IA. The affinity of NadA for both triose phosphate compounds was also very close, with a KM value of 0.64±0.12 mM for G-3P and 0.74±0.08 mM for DHAP. Thus, catalytic efficiencies were very similar (Table 1). In conclusion, both triose phosphates can be used to form QA, with G-3P performing as well as DHAP in the presence of IA. These results confirmed those of labeling experiments performed with NadA and NadB enriched extracts supplied with L-aspartate and 14C-DHAP or 14C-G-3P, where similar amounts of radioactive QA were detected18.
Specific TPI activity [µmol /min/mg]
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Figure 1. TPI activity of commercial TPI, E. coli NadA and T. maritima NadA. Rabbit muscle TPI (1-5 nM), E. coli NadA (5-10 µM) or T. maritima NadA (20 µM) were mixed with NADH (0.24 mM), G-3P (4 mM) and GPDH (1 µM) in 0.1 M TEA pH 7.6 in aerobic conditions at 37 °C (maintained using a water bath). The initial rate of the reaction (black) was monitored at 340 nm for 100 sec with a spectrophotometer. For the reverse reaction (light gray) proteins were added to NAD (1 mM), potassium arsenate (0.5 mM), DHAP (4 mM) and GAPDH (0.7 µM). For inhibition studies PGA (1 mM) was added (G-3P substrate: dark gray; DHAP substrate: white) (n≥3). (B) Inhibition of E. coli NadA QA-formation by titrated PGA. E. coli NadA (10 µM) was incubated anaerobically for 20 min at 37 °C with DHAP (2 mM) (black) or G-3P (white), OAA (5 mM), AS (10 mM) and PGA (05 mM) in 0.1 M Hepes pH 7.5, 50 mM KCl (n=13). However, when the experiment was repeated using apo-NadA, a 50% drop in activity was observed (0.5 vs 0.89 µmol/min/mg for DHAP formation and 0.04 vs 0.09 µmol/min/mg for G-3P formation) (Fig. S3B in SI), indicating that the Fe/S cluster of NadA (both Fe4S4 and Fe2S2) might play a significant structural role in NadA which could affect the TPI activity. To eliminate any possible contamination, we performed two experiments: 1) a mass spectrometry-based proteomics analysis of purified E. coli NadA; 2) measurement of TPI activity with purified NadA from a thermophilic organism, T. maritima. Purification of this enzyme involved a heating step. The E. coli TPI, TpiA, was not detected by mass spectrometry analysis of purified NadA (Table S1 and Figure S6 in SI) and T. maritima NadA was shown to display TPI activity in the same order of magnitude as that for E. coli NadA, once again displaying a thermodynamically favorable conversion direction from G-3P to DHAP (0.24 µmol/min/mg against 0.01 µmol/min/mg for G-3P production) (Figure 1). Taken together, these results demonstrate that NadA is endowed with a TPI activity, which could explain why it can use both G-3P and DHAP along with IA as substrates to produce QA. This activity makes sense in the context of cellular metabolism where both DHAP and G-3P play key roles. During glycolysis fructose 1,6 bisphosphate is converted by fructose 1,6 bisphosphate aldolase into DHAP and G-3P. G-3P is then used for glycolysis (glucose catabolism) while DHAP is used for neoglucogenesis (glucose synthesis) (Fig. S4 in SI). The ability of NadA to use both trioses enables it to ensure the synthesis of QA, and thus NAD even if one of the two trioses is not available. These results revealed a new activity of NadA, but they do not provide any information as to which molecule, DHAP or G-3P, condenses with IA to form QA, i.e, whether mechanism A or B is correct. Therefore, in subsequent experiments we used phosphoglycolic acid (PGA), an
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Biochemistry
inhibitor of TPI activity24. PGA is a transition state analog of TPI which is one atom shorter than the substrate. It binds tightly to the protein due to its structural and charge similarities to the putative cis-enediolate-like transition state structure of the TPI catalytic active site25 and provides good inhibition at 1 mM24. First, we checked that PGA inhibits NadA TPI activity. In the presence of 1 mM PGA, the TPI activity for both E. coli and T. maritima NadA was reduced to 10% (Figure 1A). Commercial rabbit TPI was inhibited by PGA to a similar extent (Figure 1A). Thus, PGA can be used at 1 mM to significantly inhibit the TPI activity of NadA and therefore should allow us to determine whether this enzyme uses DHAP or G-3P to condense with IA. Using DHAP and IA as substrates, QA production was unchanged with 1 mM PGA, while it was slightly affected with 2 to 5 mM PGA (3% and 5% reduction respectively) (Figure 1B). In contrast, when NadA was mixed with G-3P and IA, a 60% drop in activity was observed in the presence of 1 mM PGA (90% with 5 mM PGA). Similar effects were observed with the thermophilic NadA enzyme (Fig. S5 in SI). All these results show that if G-3P is not converted to DHAP no QA can be produced, demonstrating that DHAP is the triose which condenses with IA to generate QA. These results raised the following question: how QA is produced from DHAP and IA in the presence of PGA? In other words, is the same active site used for TPI and quinolinate synthase activities? If two active sites exist (AS1 and AS2), in AS1 (TPI active site) the PGA molecule would block G-3P binding and isomerization into DHAP, thus hindering QA formation in AS2. In contrast, DHAP, even though it cannot be converted to G-3P at AS1, could react directly with IA in AS2 to produce QA (Scheme S1-A). In the case of a single active site (AS) for both activities, PGA would prevent all trioses binding and isomerization. However, the condensation product of DHAP with IA (IntA) could displace PGA to allow QA formation (Scheme S1-B). This situation could not occur with G-3P which does not condense with IA. Formation of IntA from DHAP and IA is favored for two reasons: i) condensation of DHAP with IA is thermodynamically favorable with spontaneous release of inorganic phosphate and ii) some crystallographic data support early phosphate departure14,15. Indeed, NadA 3D-structures show that there is no room in the active site to accommodate a condensation product on which the phosphate group from DHAP is still present. In conclusion, this study demonstrated for the first time that NadA exhibits a TPI activity with similar properties to other TPI enzymes. This activity was exploited to determine the mechanism used by NadA to produce QA. The formal demonstration that only DHAP reacts with IA to form QA discriminates between the two proposed mechanisms in favor of mechanism (A) (Scheme 1), with early phosphate release, which is supported by crystallographic data. This discovery paves the way for mechanistic investigations of NadA, in particular to identify intermediates and design inhibitors. ASSOCIATED CONTENT Supporting Information Material and methods and additional figures. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION
Funding Sources SODC thanks the French National Research Agency (Agence Nationale pour la Recherche) for the NADBIO contract (ANR12-BS07-0018-01) and acknowledges the partial financial support from the ARCANE Labex (ANR-11-LABX-0003-01). YC thanks the ProFi Infrastructure (ANR-10-INBS-08-01). The authors declare no competing financial interest. ACKNOWLEDGMENT We thank M. Fontecave (Collège de France) for scientific discussions.
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