Allosteric regulation and substrate activation in cytosolic nucleotidase II from Legionella pneumophila.
Cytosolic nucleotidase II (cN-II) from Legionella pneumophila (Lp) catalyzes the hydrolysis of GMP and dGMP displaying sigmoidal curves, whereas catalysis of IMP hydrolysis displayed a biphasic curve in the initial rate versus substrate concentration plots. Allosteric modulators of mammalian cN-II did not activate LpcN-II although GTP, GDP and the substrate GMP were specific activators. Crystal structures of the tetrameric LpcN-II revealed an activator-binding site at the dimer interface. A double mutation in this allosteric-binding site abolished activation, confirming the structural observations. The substrate GMP acting as an activator, partitioning between the allosteric and active site, is the basis for the sigmoidicity of the initial velocity versus GMP concentration plot. The LpcN-II tetramer showed differences in subunit organization upon activator binding that are absent in the activator-bound human cN-II structure. This is the first observation of a structural change induced by activator binding in cN-II that may be the molecular mechanism for enzyme activation.


METHODS
The coordinates and structure factors reported in this paper have been submitted to the Protein Data Bank under the accession numbers 2BDE and 4G63. The accession number of GMP complexed LpcN-II is 4OHF.
LpcN-II and LpcN-II bind by molecular sieving (View interaction) LpcN-II and LpcN-II bind by x-ray crystallography (View interaction) [Structured digital abstract was added on 5 March 2014 after original online publication].
Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore 560 064, Karnataka, India.
Bharath
B
Srinivasan
Department of Biological Sciences, Northeast Structural Genomics Consortium, Columbia University, New York, NY 10027.
Farhad
F
Forouhar
Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore 560 064, Karnataka, India.
Arpit
A
Shukla
Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore 560 064, Karnataka, India.
Chethana
C
Sampangi
Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore 560 064, Karnataka, India.
Sonia
S
Kulkarni
Department of Biological Sciences, Northeast Structural Genomics Consortium, Columbia University, New York, NY 10027.
Mariam
M
Abashidze
Department of Biological Sciences, Northeast Structural Genomics Consortium, Columbia University, New York, NY 10027.
Jayaraman
J
Seetharaman
Department of Biological Sciences, Northeast Structural Genomics Consortium, Columbia University, New York, NY 10027.
Scott
S
Lew
Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers University, Department of Biochemistry, Robert Wood Johnson Medical School, Northeast Structural Genomics Consortium, Piscataway, NJ 08854.
Lei
L
Mao
Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers University, Department of Biochemistry, Robert Wood Johnson Medical School, Northeast Structural Genomics Consortium, Piscataway, NJ 08854.
Thomas B
TB
Acton
Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers University, Department of Biochemistry, Robert Wood Johnson Medical School, Northeast Structural Genomics Consortium, Piscataway, NJ 08854.
Rong
R
Xiao
Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers University, Department of Biochemistry, Robert Wood Johnson Medical School, Northeast Structural Genomics Consortium, Piscataway, NJ 08854.
John K
JK
Everett
Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers University, Department of Biochemistry, Robert Wood Johnson Medical School, Northeast Structural Genomics Consortium, Piscataway, NJ 08854.
Gaetano T
GT
Montelione
Department of Biological Sciences, Northeast Structural Genomics Consortium, Columbia University, New York, NY 10027.
Liang
L
Tong
Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore 560 064, Karnataka, India.
Hemalatha
H
Balaram
D015720:5'-Nucleotidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000494:Allosteric Regulation; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D004789:Enzyme Activation; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D016952:Legionella pneumophila / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D013045:Species Specificity; D013379:Substrate Specificity
D001426:Bacterial Proteins; D003848:Deoxyguanine Nucleotides; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D006153:Guanosine Diphosphate; C008644:nitrophenylphosphate; D006160:Guanosine Triphosphate; C029603:deoxyguanosine triphosphate; D015720:5'-Nucleotidase
5′-nucleotidase; GMP-complexed LpcN-II structure; allostery; heterotropic activation; substrate activation