Identification of the Escherichia coliNicotinic Acid Mononucleotide Adenylyltransferase Gene

Materials.A dehydrated form of Luria-Bertani broth was purchased from Gibco BRL (Gaithersburg, Md.). Ampicillin and isopropyl-β-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-β-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.).

The blunt cloning vector pSTBlue-1, the Perfectly Blunt Cloning kit, the overexpression strain Tuner(DE3), and the T7 promoter based overexpression plasmid pET-16b(+) were purchased from Novagen (Madison, Wis.). All restriction endonucleases and T4 DNA ligase were purchased from New England Biolabs (Beverly, Mass.). Platinum Pfx DNA polymerase was purchased from Gibco BRL. Automated DNA Fluorescence Sequencing was performed at the Cornell BioResource Center.

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 DH5α 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.

The PCR to amplify the open reading frame corresponding toybeN used E. coli B genomic DNA as the template and 5′-TTA TCG ACG GTT CAT ATG AAA TCT TTA CAG GCT CTG TTT GGC-3′ (inserts an NdeI site at the start of the open reading frame) and 5′-CAG TCG CAT CAT GCC CTC GAG AAC GAC AGG TAT CAG CG-3′ (inserts an XhoI site downstream of the open reading frame) as the primer pair. The PCR product was purified by the QIAquick PCR purification kit (QIAGEN). The Novagen Perfectly Blunt Cloning kit was used to blunt the fragment ends and ligate the PCR fragment into pSTBlue-1. Plasmid DNA from eight white colonies was purified and screened for the presence of the insert. A representative insert-containing plasmid was designated pCLK1039. The open reading frame was then removed from pCLK1039 by digestion with NdeI and XhoI and was ligated into similarly digested pET-16b. Colonies were screened for the presence of the insert and a representative insert-containing plasmid was designated pCLK1040. The PCR-derived DNA was sequenced and found to contain three silent mutations when compared to the database sequence for E. coliK-12.

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.

In a competition reaction containing 1.5 mM NAMN and 1.5 mM NMN, only NAAD is formed. This demonstrates that NAMN is adenylated more than 20 times faster than NMN (Fig. 1D and E). This result stands in contrast to the adenylation catalyzed by E. coli NadR, a bifunctional protein that regulates NAD biosynthesis and catalyzes the adenylation of NMN 170 times faster than the adenylation of NAMN (7).

Conclusions. Our experiments demonstrate that YbeN catalyzes the reversible adenylation of NAMN to give NAAD (Fig. 1A). We therefore propose that ybeN be renamed nadD.