Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
23
pubmed:dateCreated
2004-6-8
pubmed:abstractText
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.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jun
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
15
pubmed:volume
43
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
7610-7
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
2004
pubmed:articleTitle
Characterization of Mycobacterium tuberculosis NAD kinase: functional analysis of the full-length enzyme by site-directed mutagenesis.
pubmed:affiliation
Istituto di Biotecnologie Biochimiche, Università Politecnica delle Marche, Via Ranieri, 60131 Ancona, Italy.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't