Characterization of Mycobacterium tuberculosis NAD kinase: functional analysis of the full-length enzyme by site-directed mutagenesis.
NAD kinase is the only known enzyme catalyzing the formation of NADP, a coenzyme implicated in most reductive biosynthetic reactions and in many antioxidant defense systems. Despite its importance, nothing is known regarding its structure or mechanism of catalysis. Mycobacterium tuberculosis NAD kinase has been overexpressed in Escherichia coli and purified to homogeneity. The molecular and kinetic properties of the enzyme resulted in significant differences from those reported by others on a proteolytically degraded form of the protein. Indeed the full-length enzyme displays an allosteric behavior and shows a strict preference for inorganic polyphosphate as the phosphate donor. It is inhibited by the reaction product NADP and by both NADH and NADPH. The mycobacterial enzyme shares with all other known NAD kinases a highly conserved region (spanning residues 189-210), particularly rich in glycines, which differs from the primary sequences of all previously identified nucleotide-binding sites. Alanine-scanning mutagenesis performed on 11 conserved residues within this domain revealed its importance in catalysis. A total of 6 of 11 mutated proteins completely lost the enzymatic activity while retaining the same oligomeric state of the wild-type protein, as demonstrated by gel-filtration analysis. Substitutions of S199 and G208 with alanine rendered enzyme versions with reduced activity. Their kinetic characterization, performed on purified proteins, revealed kinetic parameters toward ATP and polyphosphate similar to those of the wild-type enzyme. On the contrary, when the kinetic analysis was performed by using NAD as the variable substrate, significant differences were observed with respect to both the allosteric behavior and the catalytic efficiency, suggesting that the mutated region is likely involved in NAD binding.
Raffaelli
Nadia
N
Istituto di Biotecnologie Biochimiche, Università Politecnica delle Marche, Via Ranieri, 60131 Ancona, Italy.
Finaurini
Lucia
L
Mazzola
Francesca
F
Pucci
Lisa
L
Sorci
Leonardo
L
Amici
Adolfo
A
Magni
Giulio
G
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D004926:Escherichia coli; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D009243:NAD / Q000494:pharmacology; D009249:NADP / Q000494:pharmacology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment
D011994:Recombinant Proteins; D009243:NAD; D009249:NADP; D017853:Phosphotransferases (Alcohol Group Acceptor); C021844:NAD kinase