Structural and biochemical studies identify tobacco SABP2 as a methyl salicylate esterase and implicate it in plant innate immunity.

Salicylic acid (SA) is a critical signal for the activation of plant defense responses against pathogen infections. We recently identified SA-binding protein 2 (SABP2) from tobacco as a protein that displays high affinity for SA and plays a crucial role in the activation of systemic acquired resistance to plant pathogens. Here we report the crystal structures of SABP2, alone and in complex with SA at up to 2.1-A resolution. The structures confirm that SABP2 is a member of the alpha/beta hydrolase superfamily of enzymes, with Ser-81, His-238, and Asp-210 as the catalytic triad. SA is bound in the active site and is completely shielded from the solvent, consistent with the high affinity of this compound for SABP2. Our biochemical studies reveal that SABP2 has strong esterase activity with methyl salicylate as the substrate, and that SA is a potent product inhibitor of this catalysis. Modeling of SABP2 with MeSA in the active site is consistent with all these biochemical observations. Our results suggest that SABP2 may be required to convert MeSA to SA as part of the signal transduction pathways that activate systemic acquired resistance and perhaps local defense responses as well.

[1]  B. Kunkel,et al.  Cross talk between signaling pathways in pathogen defense. , 2002, Current opinion in plant biology.

[2]  D. Klessig,et al.  Interconversion of the salicylic acid signal and its glucoside in tobacco. , 1993, The Plant journal : for cell and molecular biology.

[3]  E. Pichersky,et al.  Floral scent production in Clarkia breweri. III. Enzymatic synthesis and emission of benzenoid esters. , 1998, Plant physiology.

[4]  D. Klessig,et al.  Temperature-Dependent Induction of Salicylic Acid and Its Conjugates during the Resistance Response to Tobacco Mosaic Virus Infection. , 1992, The Plant cell.

[5]  G. Martin,et al.  Innate immunity in plants. , 2001, Current opinion in immunology.

[6]  Joel L. Sussman,et al.  The α/β hydrolase fold , 1992 .

[7]  M. Nardini,et al.  α/β Hydrolase fold enzymes : the family keeps growing , 1999 .

[8]  Jonathan D. G. Jones,et al.  Resistance gene-dependent plant defense responses. , 1996, The Plant cell.

[9]  D. Inzé,et al.  H2O2 and NO: redox signals in disease resistance , 1998 .

[10]  J. Botto,et al.  The plant cell , 2007, Plant Molecular Biology Reporter.

[11]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[12]  S. Seo,et al.  Induction of Salicylic Acid β-Glucosidase in Tobacco Leaves by Exogenous Salicylic Acid , 1995 .

[13]  H. Schwab,et al.  Mechanism of cyanogenesis: the crystal structure of hydroxynitrile lyase from Hevea brasiliensis. , 1996, Structure.

[14]  J. Braam,et al.  Cellular Localization of Arabidopsis Xyloglucan Endotransglycosylase-Related Proteins during Development and after Wind Stimulation , 1997, Plant physiology.

[15]  J. Draper,et al.  Compromising early salicylic acid accumulation delays the hypersensitive response and increases viral dispersal during lesion establishment in TMV-infected tobacco. , 1997, The Plant journal : for cell and molecular biology.

[16]  J. Glazebrook,et al.  Genes controlling expression of defense responses in Arabidopsis--2001 status. , 2001, Current opinion in plant biology.

[17]  G Jogl,et al.  COMO: a program for combined molecular replacement. , 2001, Acta crystallographica. Section D, Biological crystallography.

[18]  N. Yalpani,et al.  Partial purification and properties of an inducible uridine 5'-diphosphate-glucose-salicylic Acid glucosyltransferase from oat roots. , 1992, Plant physiology.

[19]  E. Pichersky,et al.  S-Adenosyl-L-methionine:salicylic acid carboxyl methyltransferase, an enzyme involved in floral scent production and plant defense, represents a new class of plant methyltransferases. , 1999, Archives of biochemistry and biophysics.

[20]  J. Draper Salicylate, superoxide synthesis and cell suicide in plant defence , 1997 .

[21]  E. Ward,et al.  A Central Role of Salicylic Acid in Plant Disease Resistance , 1994, Science.

[22]  W. Hendrickson Determination of macromolecular structures from anomalous diffraction of synchrotron radiation. , 1991, Science.

[23]  Feng Chen,et al.  An Arabidopsis thaliana gene for methylsalicylate biosynthesis, identified by a biochemical genomics approach, has a role in defense. , 2003, The Plant journal : for cell and molecular biology.

[24]  S. Haebel,et al.  The gene encoding polyneuridine aldehyde esterase of monoterpenoid indole alkaloid biosynthesis in plants is an ortholog of the alpha/betahydrolase super family. , 2000, European journal of biochemistry.

[25]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[26]  Jonathan D. G. Jones,et al.  Plant pathogens and integrated defence responses to infection , 2001, Nature.

[27]  X. Dong,et al.  Genetic dissection of systemic acquired resistance. , 2001, Current opinion in plant biology.

[28]  Jeffery L Dangl,et al.  Resistance gene signaling in plants--complex similarities to animal innate immunity. , 2003, Current opinion in immunology.

[29]  D E McRee,et al.  XtalView/Xfit--A versatile program for manipulating atomic coordinates and electron density. , 1999, Journal of structural biology.

[30]  U. Wobus,et al.  Sugar import and metabolism during seed development , 1997 .

[31]  K. Gruber,et al.  Reaction mechanism of hydroxynitrile lyases of the alpha/beta-hydrolase superfamily: the three-dimensional structure of the transient enzyme-substrate complex certifies the crucial role of LYS236. , 2004, The Journal of biological chemistry.

[32]  Dhirendra Kumar,et al.  High-affinity salicylic acid-binding protein 2 is required for plant innate immunity and has salicylic acid-stimulated lipase activity , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[33]  Eran Pichersky,et al.  The formation and function of plant volatiles: perfumes for pollinator attraction and defense. , 2002, Current opinion in plant biology.

[34]  D. Klessig,et al.  Identification of a Soluble, High-Affinity Salicylic Acid-Binding Protein in Tobacco , 1997, Plant physiology.

[35]  I. Raskin,et al.  Endogenous Methyl Salicylate in Pathogen-Inoculated Tobacco Plants , 1998 .

[36]  R. Dixon,et al.  Salicylic acid potentiates an agonist-dependent gain control that amplifies pathogen signals in the activation of defense mechanisms. , 1997, The Plant cell.

[37]  Martin J. Mueller,et al.  Cloning and expression of a tomato cDNA encoding a methyl jasmonate cleaving esterase. , 2004, European journal of biochemistry.

[38]  Jyoti Shah,et al.  Salicylic acid and disease resistance in plants. , 1999 .