A novel deamido-NAD+-binding site revealed by the trapped NAD-adenylate intermediate in the NAD+ synthetase structure

Rizzi, Menico; Bolognesi, M.; Coda, Alessandro

doi:10.1016/s0969-2126(98)00114-2

Cited by 46 publications

(63 citation statements)

References 40 publications

(55 reference statements)

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“…The NTD of A. aeolicus TilS has the amino acid sequence 32 SGGVDS 37 . This sequence is identical to the fingerprint sequence, SGGXDss (where X is any hydrophobic amino acid and lowercase letters are highly conserved amino acids), of the ATP-binding P-loop in the N-type ATP-PPase subfamily, such as GMP synthetase, NAD synthetase, asparagine synthetase, and argininosuccinate synthetase, which commonly adenylates a substrate and then attacks the adenylated substrate with a nucleophilic nitrogen from a second substrate (16)(17)(18)(19). Furthermore, the catalytic domain of the ATP-PPase subfamily has been shown to adopt a dinucleotide-binding fold, which indicates that the TilS NTD was derived from the N-type ATP-PPase.…”

Section: Resultsmentioning

confidence: 94%

Structural basis for lysidine formation by ATP pyrophosphatase accompanied by a lysine-specific loop and a tRNA-recognition domain

Nakanishi

Fukai

Ikeuchi

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Lysidine, a lysine-combined modified cytidine, is exclusively located at the anticodon wobble position (position 34) of eubacterial tRNA Ile 2 and not only converts the codon specificity from AUG to AUA, but also converts the aminoacylation specificity from recognition by methionyl-tRNA synthetase to that by isoleucyl-tRNA synthetase (IleRS). Here, we report the crystal structure of lysidine synthetase (TilS) from Aquifex aeolicus at 2.42-Å resolution. TilS forms a homodimer, and each subunit consists of the N-terminal dinucleotide-binding fold domain (NTD), with a characteristic central hole, and the C-terminal globular domain (CTD) connected by a long ␣-helical linker. The NTD shares striking structural similarity with the ATP-pyrophosphatase domain of GMP synthetase, which reminds us of the two-step reaction by TilS: adenylation of C34 and lysine attack on the C2 carbon. Conserved amino acid residues are clustered around the NTD central hole. Kinetic analyses of the conserved residues' mutants indicated that C34 of tRNA Ile 2 is adenylated by an ATP lying across the NTD central hole and that a lysine, which is activated at a loop appended to the NTD, nucleophilically attacks the C2 carbon from the rear. Escherichia coli TilS (called MesJ) has an additional CTD, which may recognize the tRNA Ile 2 acceptor stem. In contrast, a mutational study revealed that A. aeolicus TilS does not recognize the tRNA acceptor stem but recognizes the C29⅐G41 base pair in the anticodon stem. Thus, the two TilS enzymes discriminate tRNA Ile 2 from tRNA Met by strategies similar to that used by IleRS, but in distinct manners.crystal structure ͉ isoleucyl-tRNA synthetase ͉ mutagenesis ͉ RNA modification

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Section: Resultsmentioning

confidence: 94%

Structural basis for lysidine formation by ATP pyrophosphatase accompanied by a lysine-specific loop and a tRNA-recognition domain

Nakanishi

Fukai

Ikeuchi

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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“…Addition of NaAD ϩ accelerates the glutaminase activity of all of these mutants 2-to 7-fold but not to the degree observed for wild type (30-fold in specific activity terms). The D593A mutation, which corresponds to an Asp involved in NaAD ϩ binding in both Bacillus subtilis and E. coli NAD ϩ synthetases (Asp-220 and Asp-223, respectively) (17,18), results in an increase of the basal glutaminase activity. Thus, this charge-neutralizing substitution in Saccharomyces cerevisiae Qns1 may cause structural adjustments similar to those induced by NaAD ϩ binding.…”

Section: Six Classes Of Mutants In the Qns1 Ammonia Channel Alter Coomentioning

confidence: 99%

Glutamine-dependent NAD+ Synthetase

Wójcik

Seidle

Bieganowski

et al. 2006

Journal of Biological Chemistry

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Glutamine-dependent NAD؉ synthetase, Qns1, utilizes a glutamine aminotransferase domain to supply ammonia for amidation of nicotinic acid adenine dinucleotide (NaAD ؉ ) to NAD ؉ . Earlier characterization of Qns1 suggested that glutamine consumption exceeds NAD ؉ production by 40%. To explore whether Qns1 is systematically wasteful or whether additional features account for this behavior, we performed a careful kinetic and molecular genetic analysis. In fact, Qns1 possesses remarkable properties to reduce waste. The glutaminase active site is stimulated by NaAD ؉ more than 50-fold such that glutamine is not appreciably consumed in the absence of NaAD ؉ . Glutamine consumption exceeds NAD؉ production over the whole range of glutamine and NaAD ؉ substrate concentrations with greatest efficiency occurring at saturation of both substrates. Kinetic data coupled with site-directed mutagenesis of amino acids in the predicted ammonia channel indicate that NaAD ؉ stimulates the glutaminase active site in the k cat term by a synergistic mechanism that does not require ammonia utilization by the NaAD ؉ substrate. Six distinct classes of Qns1 mutants that fall within the glutaminase domain and the synthetase domain selectively inhibit components of the coordinated reaction.Glutamine-dependent NAD ϩ synthetase Qns1 (1) is one of many enzymes that couples a glutamine amidotransferase (GAT) 2 domain to a second active site that requires ammonia gas as a reactant (2-4). The second active site of Qns1 is an NAD ϩ synthetase domain that reacts nicotinic acid adenine dinucleotide (NaAD ϩ ) with ATP to form an activated, adenylylated intermediate (NaAD-AMP), releasing pyrophosphate. In turn, NaAD-AMP is attacked by ammonia to produce NAD ϩ plus AMP. Ideally, the enzyme would produce one equivalent of NAD ϩ and AMP for every glutamine hydrolyzed to glutamate. This ideal stoichiometry, depicted in Fig. 1, has been attributed to glutamine-dependent NAD ϩ synthetase since the first description of the enzyme by Preiss and Handler (5). The simplicity of Fig. 1 notwithstanding, characterization of the native (6) and recombinant (1) purified enzyme has never been entirely consistent with ideality. Whether the enzyme is, in fact, capable of perfect stoichiometry, potentially at a "sweet spot" of substrate concentrations, has not been investigated.Though eukaryotic and some prokaryotic NAD ϩ synthetases have long been understood to be glutamine-dependent enzymes (5, 7), the GAT domain of these enzymes initially escaped recognition as a glutaminase because it is neither a triad-type GAT, an N-terminal nucleophile-type GAT (2), nor one related to aminoacyl-tRNA transamidases (8). In the course of characterizing the domain structure of members of the nitrilase superfamily, we observed that the glutamine dependence of all eukaryotic and particular prokaryotic NAD ϩ synthetases could be accounted for by the presence of a putative GAT domain related to nitrilase (9, 10). Site-directed mutagenesis of yeast Qns1 coupled to biochemical and physiologi...

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“…Thus, in advance of a crystal structure of Qns1, we provide genetic evidence for an obligate intramolecular route for ammonia in Qns1 multimers. Structural Phylogenetic Excavation of the Qns1 Ammonia Channel-The crystal structure of worm NitFhit (28) and crystal structures of ammonia-dependent NAD ϩ synthetase from B. subtilis (30,31) were used to construct a low resolutionpredictive view of the organization of the putative Qns1 tetramer. Because the Nit tetramer has pairs of C termini directed away from its core structure and bacterial NAD ϩ synthetase dimers have a pair of N termini that emerge from one specific face, there is a uniquely reasonable way to associate pairs of bacterial NAD synthetase dimers with a Nit tetramer.…”

Section: Fig 3 Qns1 Contains Two Essential Active Sites For Nad ؉ Smentioning

confidence: 99%

“…The glutaminase domain of Qns1 was threaded against the 15% identical Nit tetrameric core domain of NitFhit. The NAD ϩ synthetase domain of Qns1 was threaded against the 25% identical dimeric ammonia-dependent NAD ϩ synthetase from B. subtilis bound to the crystallographically determined NaAD-AMP intermediate (31). Pairs of Nit-related C termini projected toward pairs of NAD ϩ synthetase N termini and suggested the overall organization of the domains.…”

Section: Fig 3 Qns1 Contains Two Essential Active Sites For Nad ؉ Smentioning

confidence: 99%

Eukaryotic NAD+ Synthetase Qns1 Contains an Essential, Obligate Intramolecular Thiol Glutamine Amidotransferase Domain Related to Nitrilase

Bieganowski¹,

Pace²,

Brenner³

2003

Journal of Biological Chemistry

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NAD ؉ is an essential co-enzyme for redox reactions and is consumed in lysine deacetylation and poly(ADPribosyl)ation. NAD ؉ synthetase catalyzes the final step in NAD ؉ synthesis in the well characterized de novo, salvage, and import pathways. It has been long known that eukaryotic NAD ؉ synthetases use glutamine to amidate nicotinic acid adenine dinucleotide while many purified prokaryotic NAD ؉ synthetases are ammoniadependent. Earlier, we discovered that glutamine-dependent NAD ؉ synthetases contain N-terminal domains that are members of the nitrilase superfamily and hypothesized that these domains function as glutamine amidotransferases for the associated synthetases. Here we show yeast glutamine-dependent NAD ؉ synthetase Qns1 requires both the nitrilase-related active-site residues and the NAD ؉ synthetase active-site residues for function in vivo. Despite failure to complement the lethal phenotype of qns1 disruption, the former mutants retain ammonia-dependent NAD ؉ synthetase activity in vitro, whereas the latter mutants retain basal glutaminase activity. Moreover, the two classes of mutants fail to trans-complement despite forming a stable heteromultimer in vivo. These data indicate that the nitrilaserelated domain in Qns1 is the fourth independently evolved glutamine amidotransferase domain to have been identified in nature and that glutamine-dependence is an obligate phenomenon involving intramolecular transfer of ammonia over a predicted distance of 46 Å from one active site to another within Qns1 monomers.Nicotinamide-adenine dinucleotide (NAD ϩ ) 1 and its phosphorylated form NADP are essential for oxidizing reactions in the cell, whereas reduced forms of these co-enzymes, NADH and NADPH, are essential for supplying reducing equivalents. Beyond the reversible functions of NAD ϩ in hundreds of enzymedependent redox reactions, at least two types of enzymes consume NAD ϩ in eukaryotic nuclei in the process of forming or reversing post-translational modifications. Poly(ADP-ribose) polymerases respond to DNA strand breaks by transferring the ADP-ribose moiety of NAD ϩ to target proteins, thereby facilitating DNA repair and other processes (1, 2). Sirtuins, a family of protein lysine-deacetylases related to yeast Sir2, reverse regulatory acetyl modification of lysines on histones, p53, and other proteins typically to alter the assembly of nucleoprotein complexes (3, 4). NAD ϩ is consumed by Sir2 to produce a mixture of 2Ј-and 3Ј-O-acetylated ADP-ribose plus nicotinamide and the deacetylated polypeptide (5). NAD ϩ -dependent deacetylation reactions are required not only for alterations in gene expression but also for repression of ribosomal DNA recombination and extension of lifespan in response to calorie restriction (6, 7). In prokaryotes, Sirtuins perform lysine deacetylation to alter chromatin structure (8) as well as to regulate acetyl-CoA synthetase and potentially other metabolic enzymes (9).Interest in the biological functions of Sir2 and Sirtuins has lead to re-examination of the biosynthetic ro...

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A novel deamido-NAD+-binding site revealed by the trapped NAD-adenylate intermediate in the NAD+ synthetase structure

Cited by 46 publications

References 40 publications

Structural basis for lysidine formation by ATP pyrophosphatase accompanied by a lysine-specific loop and a tRNA-recognition domain

Structural basis for lysidine formation by ATP pyrophosphatase accompanied by a lysine-specific loop and a tRNA-recognition domain

Glutamine-dependent NAD+ Synthetase

Eukaryotic NAD+ Synthetase Qns1 Contains an Essential, Obligate Intramolecular Thiol Glutamine Amidotransferase Domain Related to Nitrilase

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