Functional proteomic and structural insights into molecular recognition in the nitrilase family enzymes.

Nitrilases are a large and diverse family of nonpeptidic C-N hydrolases. The mammalian genome encodes eight nitrilase enzymes, several of which remain poorly characterized. Prominent among these are nitrilase-1 (Nit1) and nitrilase-2 (Nit2), which, despite having been shown to exert effects on cell growth and possibly serving as tumor suppressor genes, are without known substrates or selective inhibitors. In previous studies, we identified several nitrilases, including Nit1 and Nit2, as targets for dipeptide-chloroacetamide activity-based proteomics probes. Here, we have used these probes, in combination with high-resolution crystallography and molecular modeling, to systematically map the active site of Nit2 and identify residues involved in molecular recognition. We report the 1.4 A crystal structure of mouse Nit2 and use this structure to identify residues that discriminate probe labeling between the Nit1 and Nit2 enzymes. Interestingly, some of these residues are conserved across all vertebrate Nit2 enzymes and, conversely, not found in any vertebrate Nit1 enzymes, suggesting that they are key discriminators of molecular recognition between these otherwise highly homologous enzymes. Our findings thus point to a limited set of active site residues that establish distinct patterns of molecular recognition among nitrilases and provide chemical probes to selectively perturb the function of these enzymes in biological systems.

[1]  B. Maras,et al.  Is pantetheinase the actual identity of mouse and human vanin‐1 proteins? , 1999, FEBS letters.

[2]  B. Cravatt,et al.  Activity-based protein profiling: from enzyme chemistry to proteomic chemistry. , 2008, Annual review of biochemistry.

[3]  L. Lally The CCP 4 Suite — Computer programs for protein crystallography , 1998 .

[4]  D. Iliopoulos,et al.  Biological Functions of Mammalian Nit1, the Counterpart of the Invertebrate NitFhit Rosetta Stone Protein, a Possible Tumor Suppressor* , 2006, Journal of Biological Chemistry.

[5]  C. Brenner,et al.  The nitrilase superfamily: classification, structure and function , 2001, Genome Biology.

[6]  M F Sanner,et al.  Python: a programming language for software integration and development. , 1999, Journal of molecular graphics & modelling.

[7]  Matthew Bogyo,et al.  Identification of proteases that regulate erythrocyte rupture by the malaria parasite Plasmodium falciparum. , 2008, Nature chemical biology.

[8]  B. Cravatt,et al.  Mechanism‐Based Profiling of Enzyme Families , 2006 .

[9]  D S Goodsell,et al.  Automated docking of flexible ligands: Applications of autodock , 1996, Journal of molecular recognition : JMR.

[10]  A G Murzin,et al.  SCOP: a structural classification of proteins database for the investigation of sequences and structures. , 1995, Journal of molecular biology.

[11]  P. Bork,et al.  A new family of carbon‐nitrogen hydrolases , 1994, Protein science : a publication of the Protein Society.

[12]  B. Cravatt,et al.  Discovering disease-associated enzymes by proteome reactivity profiling. , 2004, Chemistry & biology.

[13]  C. Brenner,et al.  The Reported Human NADsyn2 Is Ammonia-dependent NAD Synthetase from a Pseudomonad* , 2003, Journal of Biological Chemistry.

[14]  Sherry L. Niessen,et al.  Proteomic profiling of metalloprotease activities with cocktails of active-site probes , 2006, Nature chemical biology.

[15]  Dong-Eun Kim,et al.  A Determinant Residue of Substrate Specificity in Nitrilase from Rhodococcus rhodochrous ATCC 33278 for Aliphatic and Aromatic Nitriles , 2008 .

[16]  C. Brenner Catalysis in the nitrilase superfamily. , 2002, Current opinion in structural biology.

[17]  N. Ariel,et al.  The 'aromatic patch' of three proximal residues in the human acetylcholinesterase active centre allows for versatile interaction modes with inhibitors. , 1998, The Biochemical journal.

[18]  R. Tata,et al.  Support for a three-dimensional structure predicting a Cys-Glu-Lys catalytic triad for Pseudomonas aeruginosa amidase comes from site-directed mutagenesis and mutations altering substrate specificity. , 2002, The Biochemical journal.

[19]  G. Murshudov,et al.  Refinement of macromolecular structures by the maximum-likelihood method. , 1997, Acta crystallographica. Section D, Biological crystallography.

[20]  I. Tanaka,et al.  Crystal structure of hypothetical protein PH0642 from Pyrococcus horikoshii at 1.6Å resolution , 2004, Proteins.

[21]  David S. Goodsell,et al.  Distributed automated docking of flexible ligands to proteins: Parallel applications of AutoDock 2.4 , 1996, J. Comput. Aided Mol. Des..

[22]  Z. Otwinowski,et al.  Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[23]  Rodrigo Lopez,et al.  Multiple sequence alignment with the Clustal series of programs , 2003, Nucleic Acids Res..

[24]  B. Cravatt,et al.  Activity-based protein profiling: the serine hydrolases. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[25]  C. Sander,et al.  Protein structure comparison by alignment of distance matrices. , 1993, Journal of molecular biology.

[26]  J. Piškur,et al.  The crystal structure of beta-alanine synthase from Drosophila melanogaster reveals a homooctameric helical turn-like assembly. , 2008, Journal of molecular biology.

[27]  F. Studier,et al.  Crystal structure of a putative CN hydrolase from yeast , 2003, Proteins.

[28]  W. Hsu,et al.  CRYSTAL STRUCTURE ANALYSIS OF N-CARBAMoYL-D-AMINO-ACID AMIDOHYDROLASE , 2001 .

[29]  Jay Painter,et al.  Electronic Reprint Biological Crystallography Optimal Description of a Protein Structure in Terms of Multiple Groups Undergoing Tls Motion Biological Crystallography Optimal Description of a Protein Structure in Terms of Multiple Groups Undergoing Tls Motion , 2005 .

[30]  A. Saghatelian,et al.  An enzyme that regulates ether lipid signaling pathways in cancer annotated by multidimensional profiling. , 2006, Chemistry & biology.

[31]  Y. Pekarsky,et al.  Crystal structure of the worm NitFhit Rosetta Stone protein reveals a Nit tetramer binding two Fhit dimers , 2000, Current Biology.

[32]  B. Cravatt,et al.  Substrate mimicry in an activity-based probe that targets the nitrilase family of enzymes. , 2006, Angewandte Chemie.

[33]  C. Chien,et al.  Growth inhibitory effect of the human NIT2 gene and its allelic imbalance in cancers , 2007, The FEBS journal.

[34]  A. V. van Kuilenburg,et al.  beta-Ureidopropionase deficiency: an inborn error of pyrimidine degradation associated with neurological abnormalities. , 2004, Human molecular genetics.

[35]  R J Read,et al.  Pushing the boundaries of molecular replacement with maximum likelihood. , 2003, Acta crystallographica. Section D, Biological crystallography.

[36]  A. Burlingame,et al.  Chemical Approaches for Functionally Probing the Proteome* , 2002, Molecular & Cellular Proteomics.

[37]  C. Brenner,et al.  Eukaryotic NAD+ Synthetase Qns1 Contains an Essential, Obligate Intramolecular Thiol Glutamine Amidotransferase Domain Related to Nitrilase* , 2003, Journal of Biological Chemistry.

[38]  B. Maras,et al.  Pantetheinase activity of membrane‐bound Vanin‐1: lack of free cysteamine in tissues of Vanin‐1 deficient mice , 2000, FEBS letters.