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Structural and Kinetic Characterization of NAD Synthetase from H. sapiens and M. tuberculosis
Author(s) -
Chuenchor Watchalee,
Chang Kaiti,
Doukov Tzanko I.,
Gerratana Barbara
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.963.4
Subject(s) - nad+ kinase , biochemistry , enzyme , stereochemistry , chemistry , biology
NAD synthetase catalyzes the ATP‐dependent NAD formation from nicotinic acid adenine dinucleotide in NAD biosynthesis. This enzyme is essential for survival of Mycobacterium tuberculosis but not of humans. In this work, we are determining structural and mechanistic differences between NAD synthetase from M. tuberculosis (NAD synthetase TB ) and from Homo sapiens (NAD synthetase HS ) using x‐ray crystallography and kinetic analysis. NAD synthetase TB is homooctameric with a 40 Å long inter‐subunit ammonia tunnel that allows transport of ammonia from the glutaminase active site to the synthetase active site. Structural complexes of NAD synthetase TB with ligands have been solved within a 2.6–3.0 Å resolution, revealing key amino acid residues responsible for substrate binding and catalysis. In addition, NAD synthetase HS expressed in Sf9 insect cells was co‐crystallized with an intermediate analog for structural comparison. This structure solved at a 3.0 Å resolution using molecular replacement and kinetic characterization are here reported. The crystal structures and kinetic parameters of both NAD synthetase HS and NAD synthetase TB can help clarify the basis of substrate recognition and specificity, leading to the design of high affinity inhibitors specifically targeting NAD synthetase TB .