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The cofactors flavin adenine dinucleotide, coenzyme A, and NAD(P) are all adenylated. However, the genes coding for the corresponding adenylyltransferases have been difficult to identify because the adenylated reaction products are not taken up by bacteria; therefore, auxanographic screening of mutants is impossible. The flavin adenine dinucleotide synthetase gene (ribF) and the phosphopantetheine adenylyltransferase gene (kdtB or coaD) were identified by using sequence information derived from the purified proteins (5; K. Kitatsuji, S. Ishino, S. Teshiba, and M. Arimoto, 1993, European patent application). While the Escherichia coli nicotinic acid mononucleotide (NAMN) adenylyltransferase has been partially purified (3), the corresponding gene (nadD) has not yet been identified in any microorganism (Fig. 1A). The gene for the closely related enzyme, nicotinamide mononucleotide (NMN) adenylyltransferase, involved in the salvage of NMN, has been cloned from several sources (4, 7-11).

View larger version (17K): [in this window] [in a new window]   FIG. 1.   (A) The YbeN-catalyzed adenylation reaction. (B and C) HPLC analyses of the YbeN-catalyzed adenylation reactions: (B) 1.5 mM NAMN, reaction time is 15 min; (C) 1.5 mM NMN, reaction time is 40 min. (D and E) HPLC analysis of the competitive adenylation of NAMN and NMN: (D) the reaction mixture at t = 5 min; (E) the reaction mixture at t = 0 min. Labeled peaks: 1, NAMN; 2, NMN; 3, ATP; 4, NAAD; 5, NAD.

Mutations in nadD have been previously identified in Salmonella typhimurium as 6-amino-nicotinamide-resistant mutants which had elevated levels of intracellular NAMN and reduced levels of NAMN adenylyltransferase activity (6). Most of these mutants were temperature-sensitive lethal mutants, and all mapped between lip and leuS. There are four unidentified open reading frames in this region of the E. coli genome, one of which, ybeN, had sequence homology with Rv2421c from Mycobacterium tuberculosis. Rv2421c had low sequence homology to the recently identified NMN adenylyltransferase (YLR328W) from Saccharomyces cerevisiae (4). All three genes contained a good match for the nucleotidyl transferase consensus sequence (GXFXXXHXGH) (2), which suggested that ybeN was a reasonable candidate for the NAMN adenylyltransferase. Surprisingly, a BLAST search indicated that YbeN did not show strong sequence similarity to other bacterial gene products. In this communication, we report the overexpression of ybeN and the demonstration that the gene product catalyzes the adenylation of NAMN (Fig. 1A).

Materials. A dehydrated form of Luria-Bertani broth was purchased from Gibco BRL (Gaithersburg, Md.). Ampicillin and isopropyl--D-thiogalactopyranoside (IPTG) were obtained from Jersey Lab and Glove Supply (Livingston, N.J.). Tris, agarose, EDTA, agar, dithiothreitol, 5-bromo-4-chloro-3-indolyl--D-galacto-pyranoside (X-Gal), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis molecular weight markers were from Sigma (St. Louis, Mo.). Sodium chloride was from Fisher (Pittsburgh, Pa.). NAMN, NMN, and NAD were from Aldrich-Sigma Chemical Co. (St. Louis, Mo.). Acrylamide and N,N-methylenebisacrylamide (37.5:1) were purchased from Bio-Rad Laboratories (Hercules, Calif.).

Molecular cloning. Standard methods were used for DNA restriction endonuclease digestion, ligation, and transformation (1, 12). Plasmid DNA was purified with the Wizard Plus SV DNA miniprep kit (Promega, Madison, Wis.). DNA fragments were separated by agarose gel electrophoresis, were excised, and were purified with the QIAEX II Gel Extraction Kit (QIAGEN, Valencia, Calif.). E. coli DH5 was used as a recipient for transformations during plasmid construction and for plasmid propagation and storage. A Perkin-Elmer GeneAmp PCR System 2400 was used for PCR.

Overexpression and purification of YbeN. The overexpression strain pCLK1040/Tuner(DE3) was grown in Luria-Bertani broth supplemented with 200 µg of ampicillin per ml at 37°C in a shaker at 200 rpm until the absorbance at 600 nm was approximately 0.4. The temperature was then adjusted to 15°C. Once the absorbance at 600 nm was 0.6, overexpression was induced by adding IPTG to 1.0 mM, and growth was continued for 9 h. Cells were harvested by centrifugation (6,100 × g, 30 min). Cells from 0.5 liters of media were resuspended in 30 ml of binding buffer (5 mM imidazole, 500 mM NaCl, 20 mM Tris, pH 7.9) and were lysed by a combination of lysozyme treatment and sonication (Heat Systems Ultrasonics model W-385 sonicator equipped with a 0.5-in. tip on a 5-s cycle, 50% duty for 3 min), and the lysate was cleared by centrifugation (27,000 × g, 30 min). Nickel column affinity chromatography was performed as described in the pET System Manual (Novagen). The yield of purified protein was 10 mg.

Enzymatic assays. The NAMN and NMN adenylyltransferase assays were carried out in 100 mM Tris-HCl, pH 8.0, with 2.0 mM MgCl₂. The substrates (0.25 or 1.5 mM) and enzyme (1 µg/100 µl) were incubated with 2 mM ATP at 37°C for the appropriate time interval, and 20-µl aliquots were analyzed by high-pressure liquid chromatography (HPLC). The substrate concentration for the reverse reaction was 10 mM sodium pyrophosphate and 0.1 mM NAAD. NAD was identified by comigration with a reference sample, and NAAD was purified by HPLC and identified by nuclear magnetic resonance spectroscopy.

HPLC assay. HPLC was performed on a Hewlett-Packard 1100 by using a Supelcosil LC-18-T 15-cm by 4.6-mm column (Supelco, Bellefonte, Pa.). Buffer A contained 100 mM KH₂PO₄ (aqueous), pH 7.5. Buffer B contained 80% 100 mM KH₂PO₄ (aqueous), pH 7.5, and 20% methanol. Elution conditions were 100% buffer A for 0 to 7 min, 100% buffer A to 100% buffer B in 7 to 8 min, and 100% buffer B for 8 to 13 min. Flow rate was 1 ml/min. Under these conditions, the following compounds were readily separated (retention times in parentheses): NAMN (2.08 min), NMN (2.38 min), ATP (4.54 min), NAAD (11.24 min), and NAD (11.45 min).

Enzymatic assay of YbeN. The ybeN gene product was overexpressed as a soluble 24.5-kDa protein in good yield (20 mg/liter of culture). YbeN catalyzed the complete conversion of 0.25 mM solutions of NAMN and NMN to NAAD and NAD, respectively. When the reaction was carried out using 1.5 mM NAMN, the reaction stopped when the product concentration reached 1.0 mM (Fig. 1B and C). The addition of fresh enzyme did not result in additional conversion to product; however, the reaction could be reinitiated by fourfold dilution. This suggests that NAMN adenylyltransferase is susceptible to product inhibition. NadD was able to catalyze the complete conversion of 0.1 mM NAAD to ATP and NAMN when 10 mM pyrophosphate was provided.