Herbicide-binding Sites Revealed in the Structure of Plant Acetohydroxyacid Synthase

The sulfonylureas and imidazolinones are potent commercial herbicide families. They are among the most popular choices for farmers worldwide, because they are nontoxic to animals and highly selective. These herbicides inhibit branched-chain amino acid biosynthesis in plants by targeting acetohydroxyacid synthase (AHAS, EC 2.2.1.6). This report describes the 3D structure of Arabidopsis thaliana AHAS in complex with five sulfonylureas (to 2.5 angstrom resolution) and with the imidazolinone, imazaquin (IQ; 2.8 angstrom). Neither class of molecule has a structure that mimics the substrates for the enzyme, but both inhibit by blocking a channel through which access to the active site is gained. The sulfonylureas approach within 5 angstrom of the catalytic center, which is the C2 atom of the cofactor thiamin diphosphate, whereas IQ is at least 7 angstrom from this atom. Ten of the amino acid residues that bind the sulfonylureas also bind IQ. Six additional residues interact only with the sulfonylureas, whereas there are two residues that bind IQ but not the sulfonylureas. Thus, the two classes of inhibitor occupy partially overlapping sites but adopt different modes of binding. The increasing emergence of resistant weeds due to the appearance of mutations that interfere with the inhibition of AHAS is now a worldwide problem. The structures described here provide a rational molecular basis for understanding these mutations, thus allowing more sophisticated AHAS inhibitors to be developed. There is no previously described structure for any plant protein in complex with a commercial herbicide.

Crystallization and preliminary X-ray analysis of a Kunitz-type inhibitor, textilinin-1 from Pseudonaja textilis textilis

Textilinin-1 (Txln-1), a Kunitz-type serine protease inhibitor, is a 59-amino-acid polypeptide isolated from the venom of the Australian Common Brown snake Pseudonaja textilis textilis. This molecule has been suggested as an alternative to aprotinin, also a Kunitz-type serine protease inhibitor, for use as an anti-bleeding agent in surgical procedures. Txln-1 shares only 47% amino-acid identity to aprotinin; however, six cysteine residues in the two peptides are in conserved locations. It is therefore expected that the overall fold of these molecules is similar but that they have contrasting surface features. Here, the crystallization of recombinant textilinin-1 (rTxln-1) as the free molecule and in complex with bovine trypsin (229 amino acids) is reported. Two organic solvents, phenol and 1,4-butanediol, were used as additives to facilitate the crystallization of free rTxln-1. Crystals of the rTxln-1-bovine trypsin complex diffracted to 2.0 angstrom resolution, while crystals of free rTxln-1 diffracted to 1.63 angstrom resolution.

Identification and molecular modeling of a novel, plant-like, human purple acid phosphatase

Purple acid phosphatases are a family of binuclear metallohydrolases that have been identified in plants, animals and fungi. Only one isoform of similar to 35 kDa has been isolated from animals, where it is associated with bone resorption and microbial killing through its phosphatase activity, and hydroxyl radical production, respectively. Using the sensitive PSI-BLAST search method, sequences representing new purple acid phosphatase-like proteins have been identified in mammals, insects and nematodes. These new putative isoforms are closely related to the similar to 55 kDa purple acid phosphatase characterized from plants. Secondary structure prediction of the new human isoform further confirms its similarity to a purple acid phosphatase from the red kidney bean. A structural model for the human enzyme was constructed based on the red kidney bean purple acid phosphatase structure. This model shows that the catalytic centre observed in other purple acid phosphatases is also present in this new isoform. These observations suggest that the sequences identified in this study represent a novel subfamily of plant-like purple acid phosphatases in animals and humans. (c) 2006 Elsevier B.V. All rights reserved.

A calreticulin-like protein from endoparasitoid venom fluid is involved in host hemocyte inactivation

During oviposition, most endoparasitoid wasps inject maternal factors into their hosts to interfere with host immune reactions and ensure successful development of their progeny. Since encapsulation is a major cellular defensive response of insects against intruding parasites, parasitoids have developed numerous mechanisms to suppress the host encapsulation capability by interfering with every step in the process, including recognition, adherence and spreading. In previous studies, components of Cotesia rubecula venom were shown to inhibit melanization of host hemolymph by interfering with the prophenoloxidase activation cascade and facilitate expression of polydnavirus genes. Here we report the isolation and characterization of another venom protein with similarity to calreticulin. Results indicate that C rubecula calreticulin (CrCRT) inhibits hemocyte spreading behavior, thus preventing encapsulation of the developing parasitoid. It is possible that the protein might function as an antagonist competing for binding sites with the host hemocyte calreticulin, which mediates early-encapsulation reactions. (c) 2005 Elsevier Ltd. All rights reserved.

Inhibition studies of purple acid phosphatases: Implications for the catalytic mechanism

Purple acid phosphatases (PAPs) belong to the family of binuclear metallohydrolases and catalyse the hydrolysis of a large group of phosphoester substrates at acidic pH. Despite structural conservation in their active sites PAPs appear to display mechanistic versatility. Here, aspects of the catalytic mechanism of two PAPs are investigated using the inhibitors vanadate and fluoride as probes. While the magnitude of their vanadate inhibition constants are similar the two enzymes differ with respect to the mode of inhibition; vanadate interacts in a non-competitive fashion with pig PAP (K-i = 40 mu mol L-1) while it inhibits red kidney bean PAP competitively (K-i = 30 mu mol L-1). Similarly, fluoride also acts as a competitive inhibitor for red kidney bean PAP, independent of pH, while the inhibition of pig PAP by fluoride is uncompetitive at low pH and non-competitive at higher pH, independent of metal ion composition. Furthermore, while fluoride acts as a slow-binding inhibitor in pig PAP it binds rapidly to the catalytic site of the red kidney bean enzyme. Since vanadate and fluoride are proposed to act as transition state and nucleophile mimics, respectively, the observed differences in inhibition kinetics indicate subtle but distinct variations in the reaction mechanism of these enzymes.

New era: Prophylactic surgery for patients with multiple endocrine neoplasia-2A

Background: The surgical management of patients with multiple endocrine neoplasia-2A (MEN-2A) continues to evolve with specific genotype-phenotype correlations allowing for a more tailored approach. In this study, we report the surgical management of one of the largest MEN-2A families with a rearranged during transfection (RET) codon 804 mutation. Method: This is a cohort study comprising all at-risk kindred within a single known MEN-2A family. Prophylactic total thyroidectomy with lymph node dissection was recommended to all mutation carriers aged 5 years and older. Results: There were a total of 48 at-risk individuals in the MEN-2A kindred, with 22 patients undergoing thyroidectomy after appropriate preoperative evaluation. A total of 9 patients had medullary thyroid cancer including 5 with a normal preoperative calcitonin level. A total of 11 patients had C-cell hyperplasia and 7 showed histological evidence of parathyroid disease. Only the index case had a phaeochromocytoma. Conclusion: Genetic testing for germline mutations in the RET proto-oncogene has allowed precise identification of affected RET carriers and provided the opportunity for prophylactic or 'preclinical' surgery to treat and in fact to prevent medullary thyroid cancer. This concept of prophylactic surgery based on a genetic test is likely to be applied more widely as the tools of molecular biology advance.