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Inorganic Polyphosphate/ATP-NAD kinase of Micrococcus flavus and Mycobacterium tuberculosis H37Rv
1Research Institute for Food Science, Kyoto University, Uji, Kyoto, 611-0011, Japan. kawai@food2.food.kyoto-u.ac.jp
Abstract:
An enzyme with both inorganic polyphosphate [poly(P)]- and ATP-dependent NAD kinase activities was isolated from Micrococcus flavus. The enzyme was a dimer consisting of 34 kDa subunits, and was named poly(P)/ATP-NAD kinase. Internal amino acid sequences of the enzyme showed homologies with some function-unknown proteins released on the GenBank database. Among such proteins, hypothetical Rv1695 protein (Accession No. Z98268-16), which was encoded by a gene named "Rv1695" on genomic DNA of Mycobacterium tuberculosis H37Rv, was proposed to be poly(P)-dependent NAD kinase. By cloning and expression in Escherichia coli, Rv1695 was shown to encode poly(P)/ATP-NAD kinase and named ppnk. The ppnk product, recombinant-poly(P)/ATP-NAD kinase (Ppnk) was purified and characterized. The enzyme was a tetramaer consisting of 35 kDa subunits when expressed in E. coli. Poly(P)/ATP-NAD kinases of M. flavus and Ppnk of M. tuberculosis H37Rv specifically and completely phosphorylated NAD by utilizing commercially available poly(P)s and nucleoside triphosphates as phosphoryl donors.
Insights
Researchers discovered a novel enzyme, polyphosphate/ATP-NAD kinase, from Micrococcus flavus. This enzyme, and its counterpart from Mycobacterium tuberculosis, efficiently phosphorylate NAD using polyphosphate or ATP.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Enzymes catalyzing NAD phosphorylation are crucial for cellular metabolism.
- The existence of enzymes utilizing inorganic polyphosphate [poly(P)] as a phosphoryl donor for NAD kinase activity was previously uncharacterized.
- Sequence homology suggested a potential poly(P)-dependent NAD kinase in Mycobacterium tuberculosis.
Purpose of the Study:
- To isolate and characterize an enzyme with dual polyphosphate and ATP-dependent NAD kinase activity.
- To identify and validate the gene encoding this enzyme in Mycobacterium tuberculosis.
- To elucidate the biochemical properties of the purified recombinant enzyme.
Main Methods:
- Enzyme isolation and purification from Micrococcus flavus.
- Amino acid sequencing and homology analysis against GenBank.
- Gene cloning, expression in Escherichia coli, and purification of recombinant protein.
- Biochemical characterization of enzyme kinetics and substrate specificity.
Main Results:
- An enzyme with both poly(P) and ATP-dependent NAD kinase activities was isolated from M. flavus, named poly(P)/ATP-NAD kinase.
- The hypothetical Rv1695 protein from M. tuberculosis was identified as encoding a poly(P)/ATP-NAD kinase (ppnk).
- Recombinant Ppnk from M. tuberculosis was characterized as a tetramer and demonstrated specific NAD phosphorylation using poly(P) or nucleoside triphosphates.
Conclusions:
- A novel bifunctional enzyme, poly(P)/ATP-NAD kinase, exists in M. flavus.
- The Rv1695 gene in M. tuberculosis encodes a functional poly(P)/ATP-NAD kinase (Ppnk).
- These enzymes represent a significant class of NAD-phosphorylating enzymes with broad substrate utilization.