Journal of Molecular Biology
Volume 354, Issue 2, 25 November 2005, Pages 289-303
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Crystal Structures of an NAD Kinase from Archaeoglobus fulgidus in Complex with ATP, NAD, or NADP

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NAD kinase is a ubiquitous enzyme that catalyzes the phosphorylation of NAD to NADP using ATP or inorganic polyphosphate (poly(P)) as phosphate donor, and is regarded as the only enzyme responsible for the synthesis of NADP. We present here the crystal structures of an NAD kinase from the archaeal organism Archaeoglobus fulgidus in complex with its phosphate donor ATP at 1.7 Å resolution, with its substrate NAD at 3.05 Å resolution, and with the product NADP in two different crystal forms at 2.45 Å and 2.0 Å resolution, respectively. In the ATP bound structure, the AMP portion of the ATP molecule is found to use the same binding site as the nicotinamide ribose portion of NAD/NADP in the NAD/NADP bound structures. A magnesium ion is found to be coordinated to the phosphate tail of ATP as well as to a pyrophosphate group. The conserved GGDG loop forms hydrogen bonds with the pyrophosphate group in the ATP-bound structure and the 2′ phosphate group of the NADP in the NADP-bound structures. A possible phosphate transfer mechanism is proposed on the basis of the structures presented.

Introduction

Nicotinamide adenine dinucleotide (NAD) and its phosphorylation product nicotinamide adenine dinucleotide phosphate (NADP) are two of the most important coenzymes in the cell. The importance of NAD and NADP as electron carriers in energy metabolism has long been known. Recent studies have shown that NAD and NADP are key molecules involved in a plethora of different biochemical processes, such as signal transduction, transcription, DNA repair, and detoxification reactions.1, 2, 3 The only enzyme that catalyses the phosphorylation of NAD to NADP in the presence of a phosphoryl donor, generally ATP or inorganic polyphosphate, is the ubiquitous enzyme NAD kinase (EC 2.7.1.23) (Figure 1). Since NAD kinase is the only enzyme leading to the biochemical formation of NADP, it is the key enzyme that regulates the NADP level and the NADP-dependent anabolic and biosynthetic pathways in the cell. This enzyme has been proposed to be also a potential novel antimicrobial drug target.4

To date, this enzyme has been identified, purified and characterized in bacteria, plants, vertebrates and human since its discovery several decades ago.5, 6, 7, 8, 9, 10, 11 All NAD kinases form a multimeric state in solution and divalent metals (usually magnesium) are essential for carrying out their activities. Within the characterized NAD kinases, enzymes from some bacterial organisms such as Mycobacterium tuberculosis H37Rv (Ppnk), Mycobacterium flavus and Bacillus subtilis have been shown to utilize both inorganic polyphosphate (poly(P)) and nucleoside triphosphates as phosphate donors for catalysis, and were designated as poly(P)ATP-NAD kinase,5, 7 while NAD kinases from other bacterial organisms such as Escherichia coli and higher organisms such as Saccharomyces cerevisiae and human utilize only nucleoside triphosphates as phosphate donors for catalysis and are designated as ATP-NAD kinase.6, 8, 11 It is interesting to note that the poly(P) is also proposed as an “ancient” energy carrier preceding ATP.12

All NAD kinases contain a conserved catalytic region and the NAD kinases from eukaryotic organisms have an extra N-terminal sequence. Two highly conserved and functionally important motifs have been identified within the NAD kinase family: a GGDG motif and a Gly-rich motif (Figure 2).13, 14 The role of the Gly-rich motif in NAD binding has been confirmed by mutagenesis and structural studies.13, 15, 16 The GGDG motif has been proposed to be a highly conserved motif in a super-family that includes diacylglyceride kinase, sphingosine kinase, NAD kinase and 6-phosphofructokinase.14 The 6-phosphofructokinase protein family has been studied extensively by crystallography, where the conserved GGDG motif has been observed to be involved in ADP-binding.17 This motif (SGDG in the sphingosine kinase family) is proposed to participate in nucleotide-binding in human sphingosine kinase.18 For NAD kinases, the mutation of D to A in the GGDG motif in Ppnk led to complete loss of NAD kinase activity.16 However, the mechanistic role of the conserved GGDG motif in NAD kinases has not been established.

Although NAD kinase was discovered several decades ago, its structural basis was not determined until very recently. In 2004, the crystal structures of Ppnk and its complex with NAD were determined.15, 16 These structures revealed a new structural fold involved in oligomerization of Ppnk and the NAD-binding mode. However, several key questions related to NAD kinases are still not answered. One important question is how NAD kinase binds its phosphate donor ATP or poly(P). There is very little information as to where the phosphate donor binds NAD kinase except that the GGDG motif might be involved in nucleotide substrate binding.17, 18 Here, we report four structures of an NAD kinase from the archaeal organism Archaeoglobus fulgidus (Afnk): (1) in complex with its substrate NAD (Afnk-NAD); (2) in complex with phosphate donor ATP (Afnk-ATP); and (3) in complex with the product NADP in two crystal forms (Afnk-NADP and Afnk-NADP2). The structures of this NAD kinase in complex with its three different ligands revealed detailed structural information about how NAD kinases bind the phosphate donor ATP, and how the conserved GGDG motif interacts with ATP or NADP. On the basis of the structures presented, a phosphate transfer mechanism for NAD kinases is proposed.

Section snippets

Structure determination

The structure of Afnk was first solved in an NADP-bound form. The crystals of Afnk-NADP were obtained using PEG 6 K as precipitant and they belong to space group P41212, with unit cell dimensions of a=122.1 Å, c=198.6 Å with four molecules in the asymmetric unit. The crystal structure was determined by the multi-wavelength anomalous dispersion (MAD) method using seleno-l-methionine (SeMet) substituted protein. The structure was refined at 2.45 Å resolution with Rfree=26.4% and an R-factor of 22.1%.

Discussion

The modeling of pyrophosphate (of the second ATP molecule) was based on the high-quality 1.7 Å resolution difference electron-density map, and on its coordination with the magnesium ion and the conserved GGDG motif, which had been proposed to be involved in nucleotide binding. The modeled pyrophosphate moiety was well defined in the final σA-weighted 2FoFc electron-density map. Since there was no pyrophosphate included in the protein refolding process or in the crystallization condition, we

Cloning, expression and purification

The Afnk (gi number 2650718) gene was amplified by PCR using A. fulgidus genomic DNA template and primers designed for ligation-independent cloning (LIC).20 The amplified PCR product was prepared for vector insertion by purification, quantification and treatment with phage T4 DNA polymerase (NEB) in the presence of 1 mM dTTP. The prepared insert was annealed into the LIC expression vector pB3, a derivative of pET21a (Novagen, San Diego, CA) that expresses the cloned gene with an N-terminal His6

Acknowledgements

We thank Barbara Gold for cloning, Marlene Henriquez and Bruno Martinez for expression studies and cell paste preparation, Ramona Pufan for crystallization, and John-Marc Chandonia for bioinformatics calculations. We are grateful to the staff of the Berkeley Center for Structural Biology at the Advanced Light Source (ALS). The ALS is supported by the Director, Office of Science, Office of Basic Energy Sciences, Materials Sciences Division, of the U.S. Department of Energy under contract no.

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