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 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.