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.
McDonald
Heather M
HM
Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, Birmingham, AL 35294-4440, USA.
Pruett
Pamela S
PS
Deivanayagam
Champion
C
Protasevich
Irina I
II
Carson
W Michael
WM
DeLucas
Lawrence J
LJ
Brouillette
Wayne J
WJ
Brouillette
Christie G
CG
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D019732:Amide Synthases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000586:Amination; D000595:Amino Acid Sequence; D001051:Apoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001408:Bacillus anthracis / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D015870:Gene Expression; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009525:Niacin / Q000737:chemistry / Q000378:metabolism; D010802:Phylogeny; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013379:Substrate Specificity
D001051:Apoenzymes; D009525:Niacin; D006639:Histidine; D000255:Adenosine Triphosphate; D019732:Amide Synthases; C038479:NAD+ synthase