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

• Published in: Acta crystallographica. Section D, Biological crystallography. Biological crystallography., 2007-08, Vol.63 (8), p.891-905
• Main Authors: McDonald, Heather M., Pruett, Pamela S., Deivanayagam, Champion, Protasevich, Irina I., Carson, W. Michael, DeLucas, Lawrence J., Brouillette, Wayne J., Brouillette, Christie G.
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - chemistry; Adenosine Triphosphate - metabolism; Amide Synthases - chemistry; Amide Synthases - genetics; Amide Synthases - isolation & purification; Amide Synthases - metabolism; Amination; Amino Acid Sequence; Apoenzymes - chemistry; Apoenzymes - genetics; Apoenzymes - metabolism; Bacillus anthracis; Bacillus anthracis - enzymology; Bacillus anthracis - genetics; Binding Sites; Conserved Sequence; Crystallography, X-Ray; Gene Expression; His tag; Histidine - genetics; Histidine - metabolism; Hydrogen-Ion Concentration; Models, Molecular; Molecular Sequence Data; NAD+ synthetase; Niacin - chemistry; Niacin - metabolism; Phylogeny; Protein Structure, Quaternary; Protein Structure, Tertiary; Sequence Alignment; Substrate Specificity
• ISSN: 1399-0047; 0907-4449; 1399-0047
• DOI: 10.1107/S0907444907029769

The crystal structures of NH3‐dependent NAD+ synthetase from Bacillus anthracis as the apoenzyme (1.9 Å), in complex with the natural catalytic products AMP and pyrophosphate (2.4 Å) and in complex with the substrate analog adenosine 5′‐(α,β‐methylene)triphosphate (2.0 Å) 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′‐(α,β‐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.