The Crystal Structure of E. coli Pantothenate Synthetase Confirms It as a Member of the Cytidylyltransferase Superfamily

Delft, Frank von; Lewendon, Ann; Dhanaraj, V.; Blundell, Tom L.; Abell, Chris; Smith, Alison G.

doi:10.1016/s0969-2126(01)00604-9

Cited by 73 publications

(115 citation statements)

References 48 publications

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“…This observation contradicted previous studies of the enzyme. The crystal structure of the enzyme [12] shows PS has a dimeric structure which could be consistent with the apparent behavior in solution, but no evidence for a monomerdimer or dimer-multimer equilibrium has been reported previously. However, subtraction of this dilution experiment ( Figure 1a) from a titration of PS into YfcD (Figure 1b) did provide a curve, albeit a rather featureless one (Figure 1c), and while this cannot be used to obtain robust estimates for binding constants it did suggest that the putative interaction between YfcD and PS does occur.…”

Section: Resultssupporting

confidence: 72%

Confirmation of a Protein–Protein Interaction in the Pantothenate Biosynthetic Pathway by Using Sortase‐Mediated Labelling

et al. 2016

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Abstract:High-throughput studies have been widely used to identify protein-protein interactions however the veracity of few of these candidate interactions have been demonstrated in vitro. We use a combination of isothermal titration calorimetry and fluorescence anisotropy to screen candidate interactions within the pantothenate biosynthetic pathway. In particular, we observe no interaction between the subsequent enzyme in the pathway, pantothenate synthetase (PS) and aspartate decarboxylase but do observe interaction of PS and the putative Nudix hydrolase, YfcD. Confirmation of the interaction by fluorescence anisotropy was dependent upon labelling of an adventitiously formed glycine on the protein N-terminal affinity purification tag using Sortase. Subsequent formation of the protein-protein complex led to apparent restriction of the dynamics of this tag, suggesting that this approach could be generally applied to a subset of other protein-protein interaction complexes.

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Section: Resultssupporting

confidence: 72%

Confirmation of a Protein–Protein Interaction in the Pantothenate Biosynthetic Pathway by Using Sortase‐Mediated Labelling

et al. 2016

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“…We infer that these unsuccessful trials may be due to NMN-or PP i -induced conformations that are incompatible with efficient crystal packing. NMNAT has been recognized as a member of the nucleotidyltransferase ␣/␤-phosphodiesterase superfamily, which includes class I aminoacyl-tRNA synthetase (37), luciferase (38), glycerol-3-phosphate cytidylyltransferase (39), adenylylsulfate-phosphate adenylyltransferase (40,41), ATP sulfurylase (42), phosphopantetheine adenylyltransferase (PPAT) (43), and pantothenate synthetase (44). These enzymes catalyze the transfer of a nucleotide monophosphate moiety onto different substrates through transition state stabilization without any direct involvement of the chemistry of protein residues in catalysis (18,43,45).…”

Section: Resultsmentioning

confidence: 99%

Structure of Human NMN Adenylyltransferase

Garavaglia¹,

D’Angelo²,

Emanuelli³

et al. 2002

Journal of Biological Chemistry

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Nicotinamide mononucleotide adenylyltransferase (NMNAT), a member of the nucleotidyltransferase ␣/␤-phosphodiesterases superfamily, catalyzes a universal step (NMN ؉ ATP ‫؍‬ NAD ؉ PP i ) in NAD biosynthesis. Localized within the nucleus, the activity of the human enzyme is greatly altered in tumor cells, rendering it a promising target for cancer chemotherapy. By using a combination of single isomorphous replacement and density modification techniques, the human NMNAT structure was solved by x-ray crystallography to a 2.5-Å resolution, revealing a hexamer that is composed of ␣/␤-topology subunits. The active site topology of the enzyme, analyzed through homology modeling and structural comparison with other NMNATs, yielded convincing evidence for a substrate-induced conformational change. We also observed remarkable structural conservation in the ATP-recognition motifs GXXXPX-(T/H)XXH and SXTXXR, which we take to be the universal signature for NMNATs. Structural comparison of human and prokaryotic NMNATs may also lead to the rational design of highly selective antimicrobial drugs.Beyond its pivotal role as a redox cofactor in energy transduction and cellular metabolism, NAD is also intimately involved in signaling pathways. The NAD(P) derivatives nicotinic acid adenine dinucleotide phosphate and cyclic ADP-ribose are likewise among the most potent calcium-mobilizing agents (1, 2). NAD is the substrate for poly(ADP-ribosyl)ation, a vitally important post-translation modification occurring within the nuclei of higher eukaryotes by poly(ADP-ribose) polymerase (3). NAD is also the substrate for mono-ADP-ribosyltransferase, an enzyme that transfers a single ADP-ribose unit from NAD onto target proteins in both mammalian and prokaryotic cells (4). Most recently, high levels of NAD were shown to extend yeast cell life-span, a phenomenon linked to the action of NAD-dependent catalysis of protein deacetylation (5, 6).Like ATP and GTP, NAD performs multiple tasks in both energy and signaling transduction, indicating why NAD homeostasis must be tightly regulated in all living organisms. Any impairment in NAD synthesis seriously impairs cellular metabolism, eventually resulting in cell death (7). The biosynthesis of this key redox coenzyme is accomplished either through a de novo pathway or through NAD-recycling salvage routes with notable differences between prokaryotes and eukaryotes (8). All the known biochemical pathways converge to the reaction catalyzed by NMN adenylyltransferase (EC 2.7.7.1), abbreviated NMNAT 1 (8). This transferase catalyzes the nucleophilic attack by the 5Ј phosphate NMN or nicotinic acid mononucleotide on the ␣-phosphoryl of ATP, releasing PP i and NAD or nicotinic acid adenine dinucleotide (9). Although the eukaryotic enzyme forms NAD and nicotinic acid adenine dinucleotide at similar rates, its prokaryotic counterpart prefers the deamidated substrate (nicotinic acid mononucleotide) (8). Because the reaction is fully reversible, NAD can also be reconverted to ATP (10).Human NMNAT, the only NAD/n...

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“…PPAT is a member of the nucleotidyltransferase ␣/␤ phosphodiesterase superfamily (2), which includes class I aminoacyl-tRNA synthetases (7,9,27), adenylylsulfate-phosphate adenylyltransferase (6,34), glycerol-3-phosphate cytidylyltransferase (26,35), nicotinamide mononucleotide adenylyltransferase (8), and pantothenate synthetase (33). This family is characterized by the presence of a mononucleotide binding fold and a conserved T/HXGH sequence motif, with a specific catalytic role for the second histidine within this motif.…”

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confidence: 99%

A Novel Adenylate Binding Site Confers Phosphopantetheine Adenylyltransferase Interactions with Coenzyme A

Izard

2003

J Bacteriol

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Phosphopantetheine adenylyltransferase (PPAT) regulates the key penultimate step in the essential coenzyme A (CoA) biosynthetic pathway. PPAT catalyzes the reversible transfer of an adenylyl group from Mg 2؉ :ATP to 4-phosphopantetheine to form 3-dephospho-CoA (dPCoA) and pyrophosphate. The highresolution crystal structure of PPAT complexed with CoA has been determined. Remarkably, CoA and the product dPCoA bind to the active site in distinct ways. Although the phosphate moiety within the phosphopantetheine arm overlaps, the pantetheine arm binds to the same pocket in two distinct conformations, and the adenylyl moieties of these two ligands have distinct binding sites. Moreover, the PPAT:CoA crystal structure confirms the asymmetry of binding to the two trimers within the hexameric enzyme. Specifically, the pantetheine arm of CoA bound to one protomer within the asymmetric unit displays the dPCoA-like conformation with the adenylyl moiety disordered, whereas CoA binds the twofold-related protomer in an ordered and unique fashion.Coenzyme A (CoA) is synthesized in Escherichia coli in a series of five steps utilizing pantothenate (vitamin B 5 ), cysteine, and ATP (29). Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step, the reversible transfer of an adenylyl group from ATP to 4Ј-phosphopantetheine (Ppant), yielding 3Ј-dephospho-CoA (dPCoA) and pyrophosphate (21) (Fig. 1). PPAT is the second rate-limiting step in the CoA pathway and together with the first enzyme of the pathway, pantothenate kinase, regulates the cellular CoA content (18). Ppant is derived from the metabolism of pantothenate (17), degradation of CoA (18), or the turnover of the acyl carrier protein prosthethic group (31) and exits from cells when not utilized by PPAT (18). Biochemical regulation of PPAT has not been investigated, although CoA is tightly bound to the purified enzyme (12), suggesting that, like pantothenate kinase, PPAT is feedback regulated by CoA (32).The first structure of enzymes involved in the CoA biosynthetic pathway reported was the crystal structure of E. coli PPAT in complex with dPCoA (14). To investigate the enzyme's reaction mechanism, the high-resolution crystal structures of PPAT in complex with either one of its substrates, ATP or Ppant, were subsequently determined (16). Recently, the crystal structures of E. coli pantothenate kinase in complex with 5Ј-adenylimido-diphosphate or CoA (36) and of the last enzyme in the pathway, dPCoA kinase, (from Haemophilus influenzae [23] and E. coli [24]) have also been determined. The PPAT: dPCoA crystal structure established that the enzyme displays a dinucleotide (or canonical Rossmann) binding fold (30) (Fig. 2). PPAT functions as a hexamer in solution (12), and the crystal structure is consistent with that of a hexamer having point group 32 (14). The asymmetric unit contains a dimer, and the crystallographic triad coincides with the oligomer's threefold axis. Furthermore, the PPAT:dPCoA crystal structure revealed that the PPAT hexamer consists of two...

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The Crystal Structure of E. coli Pantothenate Synthetase Confirms It as a Member of the Cytidylyltransferase Superfamily

Cited by 73 publications

References 48 publications

Confirmation of a Protein–Protein Interaction in the Pantothenate Biosynthetic Pathway by Using Sortase‐Mediated Labelling

Confirmation of a Protein–Protein Interaction in the Pantothenate Biosynthetic Pathway by Using Sortase‐Mediated Labelling

Structure of Human NMN Adenylyltransferase

A Novel Adenylate Binding Site Confers Phosphopantetheine Adenylyltransferase Interactions with Coenzyme A

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