140
S. Garavaglia and others
11 Herbert, M., Sauer, E., Smethurst, G., Kraiss, A., Hilpert, A. K. and Reidl, J. (2003)
Nicotinamide ribosyl uptake mutants in Haemophilus influenzae. Infect. Immun. 71,
5398–5401
12 Kemmer, G., Reilly, T. J., Schmidt-Brauns, J., Zlotnik, G. W., Green, B. A., Fiske, M. J.,
Herbert, M., Kraiss, A., Schlor, S., Smith, A. and Reidl, J. (2001) NadN and e (P4) are
essential for utilization of NAD and nicotinamide mononucleotide but not nicotinamide
riboside in Haemophilus influenzae. J. Bacteriol. 183, 3974–3981
13 Schmidt-Brauns, J., Herbert, M., Kemmer, G., Kraiss, A., Schlor, S. and Reidl, J. (2001) Is
a NAD pyrophosphatase activity necessary for Haemophilus influenzae type b
multiplication in the blood stream? Int. J. Med. Microbiol. 291, 219–225
14 Kurnasov, O. V., Polanuyer, B. M., Ananta, S., Sloutsky, R., Tam, A., Gerdes, S. Y. and
Osterman, A. L. (2002) Ribosylnicotinamide kinase domain of NadR protein: identification
and implications in NAD biosynthesis. J. Bacteriol. 184, 6906–6917
15 Singh, S. K., Kurnasov, O. V., Chen, B., Robinson, H., Grishin, N. V., Osterman, A. L. and
Zhang, H. (2002) Crystal structure of Haemophilus influenzae NadR protein. A
bifunctional enzyme endowed with NMN adenyltransferase and ribosylnicotinimide kinase
activities. J. Biol. Chem. 277, 33291–33299
16 Kahn, D. W. and Anderson, B. M. (1986) Characterization of Haemophilus influenzae
nucleotide pyrophosphatase. An enzyme of critical importance for growth of the organism.
J. Biol. Chem. 261, 6016–6025
17 Zagursky, R. J., Ooi, P., Jones, K. F., Fiske, M. J., Smith, R. P. and Green, B. A. (2000)
Identification of a Haemophilus influenzae 5ꢀ-nucleotidase protein: cloning of the nucA
gene and immunogenicity and characterization of the NucA protein. Infect. Immun. 68,
2525–2534
significant conformational change is a mandatory step to complete
a productive catalytic cycle. Remarkably, HiNadN recognizes
both NMN/NAD and adenosine phosphates; the integration
of biochemical and structural data suggests that adenine and
nicotinamide share the same binding pocket and demonstrates
that direct co-ordination of a phosphate group to the catalytic
Zn2 + ions is the major requirement for catalysis. The structural
investigation also led to the discovery that human CD73 is indeed
an orthologue of the bacterial enzyme and paved the way for
future investigations aiming at defining the possible physiological
role of CD73 in NAD homoeostasis in humans. The present
study provides another example of how a detailed structural
investigation of a central metabolism in bacteria can lead to the
unravelling of new functions in humans and offers a lesson in the
depth of understanding of a metabolic pathway necessary to frame
the biochemistry of NAD(P) homoeostasis in evolutionary terms.
AUTHOR CONTRIBUTION
Silvia Garavaglia and Menico Rizzi conceived the general hypothesis, designed the
experiments and analysed the results. Silvia Garavaglia, Santina Bruzzone, Antonio De
Flora and Menico Rizzi wrote the paper. Silvia Garavaglia, Laura Canella and Camilla
Cassani carried out the H. influenzae NadN overexpression, purification, crystallization,
structure determination and enzymatic characterization. Santina Bruzzone, Laura Sturla
and Elena Mannino carried out the biochemical work on human CD73. Gianna Allegrone
carried out the MS analysis and Enrico Millo performed the chemical synthesis of NR. All
authors contributed to a critical review of the paper and approved the final version prior to
submission.
18 Knofel, T. and Strater, N. (1999) X-ray structure of the Escherichia coli periplasmic
5ꢀ-nucleotidase containing a dimetal catalytic site. Nat. Struct. Biol. 6, 448–453
19 Resta, R., Yamashita, Y. and Thompson, L. F. (1998) Ecto-enzyme and signaling functions
of lymphocyte CD73. Immunol. Rev. 161, 95–109
20 Belenky, P., Bogan, K. L. and Brenner, C. (2007) NAD+ metabolism in health and disease.
Trends Biochem. Sci. 32, 12–19
21 Tempel, W., Rabeh, W. M., Bogan, K. L., Belenky, P., Wojcik, M., Seidle, H. F., Nedyalkova,
L., Yang, T., Sauve, A. A., Park, H. W. and Brenner, C. (2007) Nicotinamide riboside
kinase structures reveal new pathways to NAD+ . PLoS Biol. 5, e263
22 Belenky, P., Christensen, K. C., Gazzaniga, F., Pletnev, A. A. and Brenner, C. (2009)
Nicotinamide riboside and nicotinic acid riboside salvage in fungi and mammals.
Quantitative basis for Urh1 and purine nucleoside phosphorylase function in NAD+
metabolism. J. Biol. Chem. 284, 158–164
23 Nikiforov, A., Doelle, C., Niere, M. and Ziegler, M. (2011) Pathways and subcellular
compartmentation of NAD biosynthesis in human cells: from entry of extracellular
precursors to mitochondrial NAD generation. J. Biol. Chem. 286, 21767–21778
24 Yang, T., Chan, N. Y. and Sauve, A. A. (2007) Syntheses of nicotinamide riboside and
derivatives: effective agents for increasing nicotinamide adenine dinucleotide
concentrations in mammalian cells. J. Med. Chem. 50, 6458–6461
ACKNOWLEDGEMENTS
We thank the ESRF for data collections at the beam lines ID29 and ID14.
FUNDING
This work was supported by the Regione Piemonte (Ricerca sanitaria finalizzata and
Converging Technologies), the Italian Ministry of Education, University and Scientific
Research, the Italian Ministry of Health [grant number RF-LIG-2007-647513], the Regione
Liguria,theUniversityofGenova,theFondazioneCARIGEandtheCompagniadiSanPaolo.
25 Leslie, A. G. (1999) Integration of macromolecular diffraction data. Acta Crystallogr. Sect.
D Biol. Crystallogr. 55, 1696–1702
REFERENCES
26 Collaborative Computational Project, Number 4 (1994) The CCP4 suite: programs for
protein crystallography. Acta Crystallogr. Sect. D Biol. Crystallogr. 50, 760–763
27 McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C. and
Read, R. J. (2007) Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674
28 Perrakis, A., Morris, R. and Lamzin, V. S. (1999) Automated protein model building
combined with iterative structure refinement. Nat. Struct. Biol. 6, 458–463
29 Adams, P. D., Afonine, P. V., Bunkoczi, G., Chen, V. B., Davis, I. W., Echols, N., Headd,
J. J., Hung, L. W., Kapral, G. J., Grosse-Kunstleve, R. W. and et al. (2010) PHENIX: a
comprehensive Python-based system for macromolecular structure solution. Acta
Crystallogr. Sect. D Biol. Crystallogr. 66, 213–221
1
2
3
Turk, D.C. (1984) The pathogenicity of Haemophilus influenzae. J. Med. Microbiol. 18,
1–16
Funkhouser, A., Steinhoff, M. C. and Ward, J. (1991) Haemophilus influenzae disease and
immunization in developing countries. Rev. Infect. Dis. 13, S542–S554
Evans, N. M., Smith, D. D. and Wicken, A. J. (1974) Haemin and nicotinamide adenine
dinucleotide requirements of Haemophilus influenzae and Haemophilus parainfluenzae.
J. Med. Microbiol. 7, 359–365
4
5
6
White, D. C. and Granick, S. (1963) Hemin biosynthesis in Haemophilus. J. Bacteriol. 85,
842–850
Niven, D. F. and O’Reilly, T. (1990) Significance of V-factor dependency in the taxonomy
of Haemophilus species and related organisms. Int. J. Syst. Bacteriol. 40, 1–4
Fleischmann, R. D., Adams, M. D., White, O., Clayton, R. A., Kirkness, E. F., Kerlavage,
A. R., Bult, C. J., Tomb, J. F., Dougherty, B. A., Merrick, J. M. et al. (1995) Whole-genome
random sequencing and assembly of Haemophilus influenzae Rd. Science 269, 496–512
Reidl, J., Schlor, S., Kraiss, A., Schmidt-Brauns, J., Kemmer, G. and Soleva, E. (2000)
NADP and NAD utilization in Haemophilus influenzae. Mol. Microbiol. 35, 1573–1581
Green, B. A., Farley, J. E., Quinn-Dey, T., Deich, R. A. and Zlotnick, G. W. (1991) The e(P4)
outer membrane protein of Haemophilus influenzae: biologic activity of anti-e serum and
cloning and sequencing of the structural gene. Infect. Immun. 59, 3191–3198
Singh, H., Schuermann, J. P., Reilly, T. J., Calcutt, M. J. and Tanner, J. J. (2010)
Recognition of nucleoside monophosphate substrates by Haemophilus influenzae class C
acid phosphatase. J. Mol. Biol. 404, 639–649
30 Jones, T. A., Zou, J. Y., Cowan, S. W. and Kjeldgaard, M. (1991) Improved methods for
building protein models in electron density maps and the location of errors in these
models. Acta Crystallogr. Sect. A. Found. Crystallogr. 47, 110–119
31 Brunger, A. T. (1993) Assessment of phase accuracy by cross validation: the free R value.
Methods and applications. Acta Crystallogr. Sect. D Biol. Crystallogr. 49, 24–36
32 Guida, L., Franco, L., Zocchi, E. and De Flora, A. (1995) Structural role of disulfide bridges
in the cyclic ADP-ribose related bifunctional ectoenzyme CD38. FEBS Lett. 368, 481–484
33 Zocchi, E., Guida, L., Franco, L., Silvestro, L., Guerrini, M., Benatti, U. and De Flora, A.
(1993) Free ADP-ribose in human erythrocytes: pathways of intra-erythrocytic conversion
and non-enzymic binding to membrane proteins. Biochem. J. 295, 121–130
34 Reference deleted
7
8
9
35 Laskowski, R. A., MacArthur, M. W., Moss, D. S. and Thornton, J. M. (1993) PROCHECK:
a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr.
26, 283–291
10 Andersen, C., Maier, E., Kemmer, G., Blass, J., Hilpert, A. K., Benz, R. and Reidl, J. (2003)
Porin OmpP2 of Haemophilus influenzae shows specificity for nicotinamide-derived
nucleotide substrates. J. Biol. Chem. 278, 24269–24276
36 Knofel, T. and Strater, N. (2001) Mechanism of hydrolysis of phosphate esters by the
dimetal center of 5ꢀ-nucleotidase based on crystal structures. J. Mol. Biol. 309, 239–254
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The Authors Journal compilation 2012 Biochemical Society