Harnessing and engineering amide bond forming ligases for the synthesis of amides.

The amide functional group is ubiquitous in nature and one of the most important motifs in pharmaceuticals, agrochemicals, and other valuable products. While coupling amides and carboxylic acids is a trivial synthetic transformation, it often requires protective group manipulation, along with stoichiometric quantities of expensive and deleterious coupling reagents. Nature has evolved a range of enzymes to construct amide bonds, the vast majority of which utilize adenosine triphosphate to activate the carboxylic acid substrate for amine coupling. Despite the fact that these enzymes operate under mild conditions, as well as possessing chemoselectivity and regioselectivity that obviates the need for protecting groups, their synthetic potential has been largely unexplored. In this review, we discuss recent research into the discovery, characterization, and development of amide bond forming enzymes, with an emphasis on stand-alone ligase enzymes that can generate amides directly from simple carboxylic acid and amine substrates.

In silico and in vitro comparative activity of green tea components against Leishmania infantum.

OBJECTIVES: Green tea contains a predominant set of polyphenolic compounds with biological activities. The aim of this study was to investigate the antileishmanial activities of the main components of green tea, including catechin, (-)-epicatechin, epicatechin gallate (ECG) and (-)-epigallocatechin 3-O-gallate (EGCG), against Leishmania infantum promastigotes. METHODS: Green tea ligands and the control drug pentamidine were docked using AutoDock 4.3 software into the active sites of trypanothione synthetase and arginase, which were modelled using homology modelling programs. The colorimetric MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was used to measure L. infantum promastigotes at different concentrations of green tea compounds in a concentration- and time-dependent manner. Results were expressed as 50% and 90% inhibitory concentrations (IC50 and IC90, respectively). RESULTS: In silico and in vitro assays showed that all of the green tea compounds have antileishmanial activity. EGCG and ECG were the most active compounds against L. infantum promastigotes, with IC50 values of 27.7µM and 75µM and IC90 values of 88.4µM and 188.7µM, respectively. Pentamidine displayed greater growth inhibition than all of the other tested compounds in a concentration- and time-dependent manner. CONCLUSION: In this study, in silico and docking results were in accordance with the in vitro activity of the compounds. Moreover, EGCG and ECG showed reasonable levels of selectivity for Leishmania.

Buthionine sulfoximine is a multitarget inhibitor of trypanothione synthesis in Trypanosoma cruzi.

Buthionine sulfoximine (BSO) induces decreased glutathione (GSH) and trypanothione [T(SH)2 ] pools in trypanosomatids, presumably because only gamma-glutamylcysteine synthetase (γECS) is blocked. However, some BSO effects cannot be explained by exclusive γECS inhibition; therefore, its effect on the T(SH)2 metabolism pathway in Trypanosoma cruzi was re-examined. Parasites exposed to BSO did not synthesize T(SH)2 even when supplemented with cysteine or GSH, suggesting trypanothione synthetase (TryS) inhibition by BSO. Indeed, recombinant γECS and TryS, but not GSH synthetase, were inhibited by BSO and kinetics and docking analyses on a TcTryS 3D model suggested BSO binding at the GSH site. Furthermore, parasites overexpressing γECS and TryS showed ~ 50% decreased activities after BSO treatment. These results indicated that BSO is also an inhibitor of TryS.

Streptococcus pyogenes quinolinate-salvage pathway-structural and functional studies of quinolinate phosphoribosyl transferase and NH3 -dependent NAD+ synthetase.

Streptococcus pyogenes, also known as Group A Strep (GAS), is an obligate human pathogen that is responsible for millions of infections and numerous deaths per year. Infection manifestations can range from simple, acute pharyngitis to more complex, necrotizing fasciitis. To date, most treatments for GAS infections involve the use of common antibiotics including tetracycline and clindamycin. Unfortunately, new strains have been identified that are resistant to these drugs, therefore, new targets must be identified to treat drug-resistant strains. This work is focused on the structural and functional characterization of three proteins: spNadC, spNadD, and spNadE. These enzymes are involved in the biosynthesis of nicotinamide adenine dinucleotide (NAD+ ). The structures of spNadC and spNadE were determined. SpNadC is suggested to play a role in GAS virulence, while spNadE, functions as an NAD synthetase and is considered to be a new drug target. Determination of the spNadE structure uncovered a putative, NH3 channel, which may provide insight into the mechanistic details of NH3 -dependent NAD+ synthetases in prokaryotes. ENZYMES: Quinolinate phosphoribosyltransferase: EC2.4.2.19 and NAD synthetase: EC6.3.1.5. DATABASE: Protein structures for spNadC, spNadCΔ69A , and spNadE are deposited into Protein Data Bank under the accession codes 5HUL, 5HUO & 5HUP, and 5HUH & 5HUJ, respectively.

Discovery of novel polyamine analogs with anti-protozoal activity by computer guided drug repositioning.

Chagas disease is a parasitic infection caused by the protozoa Trypanosoma cruzi that affects about 6 million people in Latin America. Despite its sanitary importance, there are currently only two drugs available for treatment: benznidazole and nifurtimox, both exhibiting serious adverse effects and limited efficacy in the chronic stage of the disease. Polyamines are ubiquitous to all living organisms where they participate in multiple basic functions such as biosynthesis of nucleic acids and proteins, proliferation and cell differentiation. T. cruzi is auxotroph for polyamines, which are taken up from the extracellular medium by efficient transporters and, to a large extent, incorporated into trypanothione (bis-glutathionylspermidine), the major redox cosubstrate of trypanosomatids. From a 268-compound database containing polyamine analogs with and without inhibitory effect on T. cruzi we have inferred classificatory models that were later applied in a virtual screening campaign to identify anti-trypanosomal compounds among drugs already used for other therapeutic indications (i.e. computer-guided drug repositioning) compiled in the DrugBank and Sweetlead databases. Five of the candidates identified with this strategy were evaluated in cellular models from different pathogenic trypanosomatids (T. cruzi wt, T. cruzi PAT12, T. brucei and Leishmania infantum), and in vitro models of aminoacid/polyamine transport assays and trypanothione synthetase inhibition assay. Triclabendazole, sertaconazole and paroxetine displayed inhibitory effects on the proliferation of T. cruzi (epimastigotes) and the uptake of putrescine by the parasite. They also interfered with the uptake of others aminoacids and the proliferation of infective T. brucei and L. infantum (promastigotes). Trypanothione synthetase was ruled out as molecular target for the anti-parasitic activity of these compounds.

Structure and mechanism of Escherichia coli glutathionylspermidine amidase belonging to the family of cysteine; histidine-dependent amidohydrolases/peptidases.

The bifunctional Escherichia coli glutathionylspermidine synthetase/amidase (GspSA) catalyzes both the synthesis and hydrolysis of Gsp. Its amidase domain (GspA), which catalyzes the hydrolysis of Gsp into glutathione and spermidine, plays an important role in redox sensing and protein S-thiolation. To gain insight of the regulation and catalytic mechanism of and further understand the recycling of the Gsp dimer and Gsp-S-protein adducts, we solved two crystal structures of GspA and GspSA both with the C59A mutation and bound with the substrate, Gsp. In both structures, Cys59, His131, and Glu147 form the catalytic triad, which is similar to other cysteine proteases. Comparison of the GspA_Gsp complex and apo GspSA structures indicates that on binding with Gsp, the side chains of Asn149 and Gln58 of the amidase domain are induced to move closer to the carbonyl oxygen of the cleaved amide bond of Gsp, thereby participating in catalysis. In addition, the helix-loop region of GspA, corresponding to the sequence (30)YSSLDPQEYEDDA(42), involves in regulating the substrate binding. Our previous study indicated that the thiol of Cys59 of GspA is only oxidized to sulfenic acid by H(2)O(2). When comparing the active site of GspA with those of other cysteine proteases, we found that limited space and hydrophobicity of the environment around Cys59 play an important role to inhibit its further oxidation. The structural results presented here not only elucidate the catalytic mechanism and regulation of GspA but also help us to design small molecules to inhibit or probe for the activity of GspA.

Protein S-thiolation by Glutathionylspermidine (Gsp): the role of Escherichia coli Gsp synthetASE/amidase in redox regulation.

Certain bacteria synthesize glutathionylspermidine (Gsp), from GSH and spermidine. Escherichia coli Gsp synthetase/amidase (GspSA) catalyzes both the synthesis and hydrolysis of Gsp. Prior to the work reported herein, the physiological role(s) of Gsp or how the two opposing GspSA activities are regulated had not been elucidated. We report that Gsp-modified proteins from E. coli contain mixed disulfides of Gsp and protein thiols, representing a new type of post-translational modification formerly undocumented. The level of these proteins is increased by oxidative stress. We attribute the accumulation of such proteins to the selective inactivation of GspSA amidase activity. X-ray crystallography and a chemical modification study indicated that the catalytic cysteine thiol of the GspSA amidase domain is transiently inactivated by H(2)O(2) oxidation to sulfenic acid, which is stabilized by a very short hydrogen bond with a water molecule. We propose a set of reactions that explains how the levels of Gsp and Gsp S-thiolated proteins are modulated in response to oxidative stress. The hypersensitivities of GspSA and GspSA/glutaredoxin null mutants to H(2)O(2) support the idea that GspSA and glutaredoxin act synergistically to regulate the redox environment of E. coli.

The pcsA gene from Streptomyces diastaticus var. 108 encodes a polyene carboxamide synthase with broad substrate specificity for polyene amides biosynthesis.

Two structurally related polyene macrolides are produced by Streptomyces diastaticus var. 108: rimocidin (3a) and CE-108 (2a). Both bioactive metabolites are biosynthesized from the same pathway through type I polyketide synthases by choosing a starter unit either acetate or butyrate, resulting in 2a or 3a formation, respectively. Two additional polyene amides, CE-108B (2b) and rimocidin B (3b), are also produced "in vivo" when this strain was genetically modified by transformation with engineered SCP2*-derived vectors carrying the ermE gene. The two polyene amides, 2b and 3b, showed improved pharmacological properties, and are generated by a tailoring activity involved in the conversion of the exocyclic carboxylic group of 2a and 3a into their amide derivatives. The improvement on some biological properties of the resulting polyenes, compared with that of the parental compounds, encourages our interest for isolating the tailoring gene responsible for the polyene carboxamide biosynthesis, aimed to use it as tool for generating new bioactive compounds. In this work, we describe the isolation from S. diastaticus var. 108 the corresponding gene, pcsA, encoding a polyene carboxamide synthase, belonging to the Class II glutamine amidotransferases and responsible for "in vivo" and "in vitro" formation of CE-108B (2b) and rimocidin B (3b). The fermentation broth from S. diastaticus var. 108 engineered with the appropriate pcsA gene construction, showed the polyene amides to be the major bioactive compounds.

Amidoligases with ATP-grasp, glutamine synthetase-like and acetyltransferase-like domains: synthesis of novel metabolites and peptide modifications of proteins.

Recent studies have shown that the ubiquitin system had its origins in ancient cofactor/amino acid biosynthesis pathways. Preliminary studies also indicated that conjugation systems for other peptide tags on proteins, such as pupylation, have evolutionary links to cofactor/amino acid biosynthesis pathways. Following up on these observations, we systematically investigated the non-ribosomal amidoligases of the ATP-grasp, glutamine synthetase-like and acetyltransferase folds by classifying the known members and identifying novel versions. We then established their contextual connections using information from domain architectures and conserved gene neighborhoods. This showed remarkable, previously uncharacterized functional links between diverse peptide ligases, several peptidases of unrelated folds and enzymes involved in synthesis of modified amino acids. Using the network of contextual connections we were able to predict numerous novel pathways for peptide synthesis and modification, amine-utilization, secondary metabolite synthesis and potential peptide-tagging systems. One potential peptide-tagging system, which is widely distributed in bacteria, involves an ATP-grasp domain and a glutamine synthetase-like ligase, both of which are circularly permuted, an NTN-hydrolase fold peptidase and a novel alpha helical domain. Our analysis also elucidates key steps in the biosynthesis of antibiotics such as friulimicin, butirosin and bacilysin and cell surface structures such as capsular polymers and teichuronopeptides. We also report the discovery of several novel ribosomally synthesized bacterial peptide metabolites that are cyclized via amide and lactone linkages formed by ATP-grasp enzymes. We present an evolutionary scenario for the multiple convergent origins of peptide ligases in various folds and clarify the bacterial origin of eukaryotic peptide-tagging enzymes of the TTL family.

NAD(P) biosynthesis enzymes as potential targets for selective drug design.

NAD(P) biosynthetic pathways can be considered a generous source of enzymatic targets for drug development. Key reactions for NAD(P) biosynthesis in all organisms, common to both de novo and salvage routes, are catalyzed by NMN/NaMN adenylyltransferase (NMNAT), NAD synthetase (NADS), and NAD kinase (NADK). These reactions represent a three-step pathway, present in the vast majority of living organisms, which is responsible for the generation of both NAD and NADP cellular pools. The validation of these enzymes as drug targets is based on their essentiality and conservation among a large variety of pathogenic microorganisms, as well as on their differential structural features or their differential metabolic contribution to NAD(P) homeostasis between microbial and human cell types. This review describes the structural and functional properties of eubacterial and human enzymes endowed with NMNAT, NADS, and NADK activities, as well as with nicotinamide phosphoribosyltransferase (NamPRT) and nicotinamide riboside kinase (NRK) activities, highlighting the species-related differences, with emphasis on their relevance for drug design. In addition, since the overall NMNAT activity in humans is accounted by multiple isozymes differentially involved in the metabolic activation of antineoplastic compounds, their individual diagnostic value for early therapy optimization is outlined. The involvement of human NMNAT in neurodegenerative disorders and its role in neuroprotection is also discussed.

Leishmania trypanothione synthetase-amidase structure reveals a basis for regulation of conflicting synthetic and hydrolytic activities.

The bifunctional trypanothione synthetase-amidase catalyzes biosynthesis and hydrolysis of the glutathione-spermidine adduct trypanothione, the principal intracellular thiol-redox metabolite in parasitic trypanosomatids. These parasites are unique with regard to their reliance on trypanothione to determine intracellular thiol-redox balance in defense against oxidative and chemical stress and to regulate polyamine levels. Enzymes involved in trypanothione biosynthesis provide essential biological activities, and those absent from humans or for which orthologues are sufficiently distinct are attractive targets to underpin anti-parasitic drug discovery. The structure of Leishmania major trypanothione synthetase-amidase, determined in three crystal forms, reveals two catalytic domains. The N-terminal domain, a cysteine, histidine-dependent amidohydrolase/peptidase amidase, is a papain-like cysteine protease, and the C-terminal synthetase domain displays an ATP-grasp family fold common to C:N ligases. Modeling of substrates into each active site provides insight into the specificity and reactivity of this unusual enzyme, which is able to catalyze four reactions. The domain orientation is distinct from that observed in a related bacterial glutathionylspermidine synthetase. In trypanothione synthetase-amidase, the interactions formed by the C terminus, binding in and restricting access to the amidase active site, suggest that the balance of ligation and hydrolytic activity is directly influenced by the alignment of the domains with respect to each other and implicate conformational changes with amidase activity. The potential inhibitory role of the C terminus provides a mechanism to control relative levels of the critical metabolites, trypanothione, glutathionylspermidine, and spermidine in Leishmania.

Structural adaptation of an interacting non-native C-terminal helical extension revealed in the crystal structure of NAD+ synthetase from Bacillus anthracis.

The crystal structures of NH(3)-dependent NAD+ synthetase from Bacillus anthracis as the apoenzyme (1.9 A), in complex with the natural catalytic products AMP and pyrophosphate (2.4 A) and in complex with the substrate analog adenosine 5'-(alpha,beta-methylene)triphosphate (2.0 A) have been determined. NAD+ synthetase catalyzes the last step in the biosynthesis of the vitally important cofactor NAD+. In comparison to other NAD+ synthetase crystal structures, the C-terminal His-tagged end of the apoenzyme adopts a novel helical conformation, causing significant compensatory changes in the region. The structural accommodations observed in B. anthracis NAD+ synthetase are remarkable in the absence of adverse affects on enzyme activity. They also illustrate a rare example of the influence of a non-native C-terminal His-tag extension on the structure of a native protein. In contrast to the apoenzyme, when AMP and pyrophosphate or adenosine 5'-(alpha,beta-methylene)triphosphate are bound, the C-terminus adopts a conformation that allows ATP binding and overall the structure then resembles other NAD+ synthetase structures. The structures of NAD+ synthetase complexes from B. anthracis are compared with published X-ray crystal structures of the enzyme from B. subtilis, Escherichia coli and Helicobacter pylori. These comparisons support the novel observation that P1 and P2 loop ordering is not a consequence of crystal contacts but rather a consequence of intrinsic intramolecular interactions within the ordered subunit.

Dual binding sites for translocation catalysis by Escherichia coli glutathionylspermidine synthetase.

Most organisms use glutathione to regulate intracellular thiol redox balance and protect against oxidative stress; protozoa, however, utilize trypanothione for this purpose. Trypanothione biosynthesis requires ATP-dependent conjugation of glutathione (GSH) to the two terminal amino groups of spermidine by glutathionylspermidine synthetase (GspS) and trypanothione synthetase (TryS), which are considered as drug targets. GspS catalyzes the penultimate step of the biosynthesis-amide bond formation between spermidine and the glycine carboxylate of GSH. We report herein five crystal structures of Escherichia coli GspS in complex with substrate, product or inhibitor. The C-terminal of GspS belongs to the ATP-grasp superfamily with a similar fold to the human glutathione synthetase. GSH is likely phosphorylated at one of two GSH-binding sites to form an acylphosphate intermediate that then translocates to the other site for subsequent nucleophilic addition of spermidine. We also identify essential amino acids involved in the catalysis. Our results constitute the first structural information on the biochemical features of parasite homologs (including TryS) that underlie their broad specificity for polyamines.

Structures of Escherichia coli NAD synthetase with substrates and products reveal mechanistic rearrangements.

Nicotinamide adenine dinucleotide synthetases (NADS) catalyze the amidation of nicotinic acid adenine dinucleotide (NAAD) to yield the enzyme cofactor nicotinamide adenine dinucleotide (NAD). Here we describe the crystal structures of the ammonia-dependent homodimeric NADS from Escherichia coli alone and in complex with natural substrates and with the reaction product NAD. The structures disclosed two NAAD/NAD binding sites at the dimer interface and an adenosine triphosphate (ATP) binding site within each subunit. Comparison with the Bacillus subtilis NADS showed pronounced chemical differences in the NAAD/NAD binding sites and less prominent differences in the ATP binding pockets. In addition, the E. coli NADS structures revealed unexpected dynamical rearrangements in the NAAD/NAD binding pocket upon NAAD-to-NAD conversion, which define a catalysis state and a substrate/product exchange state. The two states are adopted by concerted movement of the nicotinysyl moieties of NAAD and NAD, Phe-170, and residues 224-228, which may be triggered by differential coordination of a magnesium ion to NAAD and NAD. Phylogenetic structure comparisons suggest that the present results are relevant for designing species-specific antibiotics.

Properties of trypanothione synthetase from Trypanosoma brucei.

Trypanothione [N(1),N(8)-bis(glutathionyl)spermidine] plays a central role in defence against oxidant damage, ribonucleotide metabolism and in resistance to certain drugs in trypanosomatids. In Crithidia fasciculata, synthesis of trypanothione involves sequential conjugation of two molecules of glutathione (GSH) to spermidine by two enzymes: glutathionylspermidine synthetase (GspS; EC 6.3.1.8) and trypanothione synthetase (TryS; EC 6.3.1.9), whereas in Trypanosoma cruzi both steps are catalysed by an unusual TryS with broad substrate specificity. To determine which route operates in T. brucei, we have cloned and expressed a single copy gene with similarity to C. fasciculata and T. cruzi TRYS. The purified recombinant protein catalyses formation of trypanothione from either spermidine and GSH, or glutathionylspermidine and GSH. The enzyme displays high substrate inhibition with GSH as variable substrate (apparent K(m)=56 microM, K(i)(s)=37 microM, k(cat)=2.9s(-1)). At a fixed subsaturating GSH concentration (100 microM), the enzyme obeys simple hyperbolic kinetics yielding apparent K(m) values for spermidine, glutathionylspermidine and MgATP of 38, 2.4, and 7.1 microM, respectively. Recombinant TryS can also catalyse conversion of spermine to glutathionylspermine and bis(glutathionyl)spermine, as recently reported for T. cruzi. The enzyme has amidase activity that can be inhibited by iodoacetamide. Studies using GSH and polyamine analogues identified GSH as the critical determinant for recognition by the amidase domain. Thus, the biosynthesis and degradation of trypanothione are similar in African and American trypanosomes, and different from the insect trypanosomatid, C. fasciculata.

The reported human NADsyn2 is ammonia-dependent NAD synthetase from a pseudomonad.

Nicotinamide-adenine dinucleotide (NAD+) synthetases catalyze the last step in NAD+ metabolism in the de novo, import, and salvage pathways that originate from tryptophan (or aspartic acid), nicotinic acid, and nicotinamide, respectively, and converge on nicotinic acid mononucleotide. NAD+ synthetase converts nicotinic acid adenine dinucleotide to NAD+ via an adenylylated intermediate. All of the known eukaryotic NAD+ synthetases are glutamine-dependent, hydrolyzing glutamine to glutamic acid to provide the attacking ammonia. In the prokaryotic world, some NAD+ synthetases are glutamine-dependent, whereas others can only use ammonia. Earlier, we noted a perfect correlation between presence of a domain related to nitrilase and glutamine dependence and then proved in the accompanying paper (Bieganowski, P., Pace, H. C., and Brenner, C. (2003) J. Biol. Chem. 278, 33049-33055) that the nitrilase-related domain is an essential, obligate intramolecular, thiol-dependent glutamine amidotransferase in the yeast NAD+ synthetase, Qns1. Independently, human NAD+ synthetase was cloned and shown to depend on Cys-175 for glutamine-dependent but not ammonia-dependent NAD+ synthetase activity. Additionally, it was claimed that a 275 amino acid open reading frame putatively amplified from human glioma cell line LN229 encodes a human ammonia-dependent NAD+ synthetase and this was speculated largely to mediate NAD+ synthesis in human muscle tissues. Here we establish that the so-called NADsyn2 is simply ammonia-dependent NAD+ synthetase from Pseudomonas, which is encoded on an operon with nicotinic acid phosphoribosyltransferase and, in some Pseudomonads, with nicotinamidase.

Molecular identification of human glutamine- and ammonia-dependent NAD synthetases. Carbon-nitrogen hydrolase domain confers glutamine dependency.

NAD synthetase catalyzes the final step in the biosynthesis of NAD. In the present study, we obtained cDNAs for two types of human NAD synthetase (referred as NADsyn1 and NADsyn2). Structural analysis revealed in both NADsyn1 and NADsyn2 a domain required for NAD synthesis from ammonia and in only NADsyn1 an additional carbon-nitrogen hydrolase domain shared with enzymes of the nitrilase family that cleave nitriles as well as amides to produce the corresponding acids and ammonia. Consistent with the domain structures, biochemical assays indicated (i) that both NADsyn1 and NADsyn2 have NAD synthetase activity, (ii) that NADsyn1 uses glutamine as well as ammonia as an amide donor, whereas NADsyn2 catalyzes only ammonia-dependent NAD synthesis, and (iii) that mutant NADsyn1 in which Cys-175 corresponding to the catalytic cysteine residue in nitrilases was replaced with Ser does not use glutamine. Kinetic studies suggested that glutamine and ammonia serve as physiological amide donors for NADsyn1 and NADsyn2, respectively. Both synthetases exerted catalytic activity in a multimeric form. In the mouse, NADsyn1 was seen to be abundantly expressed in the small intestine, liver, kidney, and testis but very weakly in the skeletal muscle and heart. In contrast, expression of NADsyn2 was observed in all tissues tested. Therefore, we conclude that humans have two types of NAD synthetase exhibiting different amide donor specificity and tissue distributions. The ammonia-dependent synthetase has not been found in eucaryotes until this study. Our results also indicate that the carbon-nitrogen hydrolase domain is the functional domain of NAD synthetase to make use of glutamine as an amide donor in NAD synthesis. Thus, glutamine-dependent NAD synthetase may be classified as a possible glutamine amidase in the nitrilase family. Our molecular identification of NAD synthetases may prove useful to learn more of mechanisms regulating cellular NAD metabolism.

Heterologous expression, purification, and enzymatic activity of Mycobacterium tuberculosis NAD(+) synthetase.

The enzyme NAD(+) synthetase (NadE) catalyzes the last step of NAD biosynthesis. Given NAD vital role in cell metabolism, the enzyme represents a valid target for the development of new antimycobacterial agents. In the present study we expressed and purified two putative forms of Mycobacterium tuberculosis NAD(+) synthetase, differing in the polypeptide chain length (NadE-738 and NadE-679). Furthermore, we evaluated several systems for the heterologous expression and large scale purification of the enzyme. In particular, we compared the efficiency of production, the yield of purification, and the catalytic activity of recombinant enzyme in different hosts, ranging from Escherichia coli strains to cultured High Five (Trichoplusia ni BTI-TN-5B1-4) insect cells. Among the systems assayed, we found that the expression of a thioredoxin-NadE fusion protein in E. coli Origami(DE3) is the best system in obtaining highly pure, active NAD(+) synthetase. The recombinant enzyme maintained its activity even after proteolytic cleavage of thioredoxin moiety. Biochemical evidence suggests that the shorter form (NadE-679) may be the real M. tuberculosis NAD(+) synthetase. These results enable us to obtain a purified product for structure-function analysis and high throughput assays for rapid screening of compounds which inhibit enzymatic activity.

NH3-dependent NAD+ synthetase from Bacillus subtilis at 1 A resolution.

The final step of NAD+ biosynthesis includes an amide transfer to nicotinic acid adenine dinucleotide (NaAD) catalyzed by NAD+ synthetase. This enzyme was co-crystallized in microgravity with natural substrates NaAD and ATP at pH 8.5. The crystal was exposed to ammonium ions, synchrotron diffraction data were collected and the atomic model was refined anisotropically at 1 A resolution to R = 11.63%. Both binding sites are occupied by the NAD-adenylate intermediate, pyrophosphate and two magnesium ions. The atomic resolution of the structure allows better definition of non-planar peptide groups, reveals a low mean anisotropy of protein and substrate atoms and indicates the H-atom positions of the phosphoester group of the reaction intermediate. The phosphoester group is protonated at the carbonyl O atom O7N, suggesting a carbenium-ion structure stabilized by interactions with two solvent sites presumably occupied by ammonia and a water molecule. A mechanism is proposed for the second catalytic step, which includes a nucleophilic attack by the ammonia molecule on the intermediate.