Mechanistic insights into the first Lygus-active ß-pore forming protein.

The cotton pests Lygus hesperus and Lygus lineolaris can be controlled by expressing Cry51Aa2.834_16 in cotton. Insecticidal activity of pore-forming proteins is generally associated with damage to the midgut epithelium due to pores, and their biological specificity results from a set of key determinants including proteolytic activation and receptor binding. We conducted mechanistic studies to gain insight into how the first Lygus-active ß-pore forming protein variant functions. Biophysical characterization revealed that the full-length Cry51Aa2.834_16 was a stable dimer in solution, and when exposed to Lygus saliva or to trypsin, the protein underwent proteolytic cleavage at the C-terminus of each of the subunits, resulting in dissociation of the dimer to two separate monomers. The monomer showed tight binding to a specific protein in Lygus brush border membranes, and also formed a membrane-associated oligomeric complex both in vitro and in vivo. Chemically cross-linking the ß-hairpin to the Cry51Aa2.834_16 body rendered the protein inactive, but still competent to compete for binding sites with the native protein in vivo. Our study suggests that disassociation of the Cry51Aa2.834_16 dimer into monomeric units with unoccupied head-region and sterically unhindered ß-hairpin is required for brush border membrane binding, oligomerization, and the subsequent steps leading to insect mortality.

Structural and functional characterization of triketone dioxygenase from Oryza Sativa.

The transgenic expression of rice triketone dioxygenase (TDO; also known as HIS1) can provide protection from triketone herbicides to susceptible dicot crops such as soybean. Triketones are phytotoxic inhibitors of plant hydroxyphenylpyruvate dioxygenases (HPPD). The TDO gene codes for an iron/2-oxoglutarate-dependent oxidoreductase. We obtained an X-ray crystal structure of TDO using SeMet-SAD phasing to 3.16 Å resolution. The structure reveals that TDO possesses a fold like that of Arabidopsis thaliana 2-oxoglutarate­iron-dependent oxygenase anthocyanidin synthase (ANS). Unlike ANS, this TDO structure lacks bound metals or cofactors, and we propose this is because the disordered flexible loop over the active site is sterically constrained from folding properly in the crystal lattice. A combination of mass spectrometry, nuclear magnetic resonance, and enzyme activity studies indicate that rice TDO oxidizes mesotrione in a series of steps; first producing 5-hydroxy-mesotrione and then oxy-mesotrione. Evidence suggests that 5-hydroxy-mesotrione is a much weaker inhibitor of HPPD than mesotrione, and oxy-mesotrione has virtually no inhibitory activity. Of the close homologues which have been tested, only corn and rice TDO have enzymatic activity and the ability to protect plants from mesotrione. Correlating sequence and structure has identified four amino acids necessary for TDO activity. Introducing these four amino acids imparts activity to a mesotrione-inactive TDO-like protein from sorghum, which may expand triketone herbicide resistance in new crop species.

The enzymology of alanine aminotransferase (AlaAT) isoforms from Hordeum vulgare and other organisms, and the HvAlaAT crystal structure.

In this paper we describe the expression, purification, kinetics and biophysical characterization of alanine aminotransferase (AlaAT) from the barley plant (Hordeum vulgare). This dimeric PLP-dependent enzyme is a pivotal element of several key metabolic pathways from nitrogen assimilation to carbon metabolism, and its introduction into transgenic plants results in increased yield. The enzyme exhibits a bi-bi ping-pong reaction mechanism with a K(m) for alanine, 2-oxoglutarate, glutamate and pyruvate of 3.8, 0.3, 0.8 and 0.2 mM, respectively. Barley AlaAT catalyzes the forward (alanine-forming) reaction with a k(cat) of 25.6 s(-1), the reverse (glutamate-forming) reaction with k(cat) of 12.1 s(-1) and an equilibrium constant of ~0.5. The enzyme is also able to utilize aspartate and oxaloacetate with ~10% efficiency as compared to the native substrates, which makes it much more specific than related bacterial/archaeal enzymes (that also have lower K(m) values). We have crystallized barley AlaAT in complex with PLP and l-cycloserine and solved the structure of this complex at 2.7 Å resolution. This is the first example of a plant AlaAT structure, and it reveals a canonical aminotransferase fold similar to structures of the Thermotoga maritima, Pyrococcus furiosus, and human enzymes. This structure bridges our structural understanding of AlaAT mechanism between three kingdoms of life and allows us to shed some light on the specifics of the catalysis performed by these proteins.

Phenotypic effects from the expression of a deregulated AtGAD1 transgene and GABA pathway suppression mutants in maize.

Glutamate decarboxylase (GAD; EC 4.1.1.15) catalyzes the irreversible decarboxylation of glutamate to produce γ-aminobutyric acid (GABA); a ubiquitous non-protein amino acid involved in the regulation of several aspects of plant metabolism and physiology. To study the function of GAD and GABA in maize, we have; 1) introduced native and deregulated forms of AtGAD1 into maize with the intent of increasing the synthesis of GABA and 2) introduced constructs into maize designed to suppress the activity of several GABA shunt, GABA transport and GABA pathway genes. Maize plants expressing the deregulated AtGAD1 exhibit a severe chlorosis and retarded growth phenotype and have high levels of GABA, and Ca++/CaM-independent GAD activity. Plants expressing the suppression constructs for GABA biosynthetic and transport pathway genes had no observable phenotype whereas a knockout of GABA catabolic pathway genes led to growth and developmental defects under standard growth conditions. The implications of this study to our understanding of the action and function of GABA and GAD in crops are discussed.

Phylogenetic and expression analysis of sucrose phosphate synthase isozymes in plants.

In plants and microbes, sucrose phosphate synthase (SPS) is an important enzyme in sucrose biosynthesis. Several different isozymes of SPS exist in plants. Genomic and EST sequence data from Arabidopsis, rice and maize has been analyzed. This analysis has revealed that the Arabidopsis genome contains four unique SPS genes. The rice databases (Monsanto proprietary, and public databases) contain five unique full-length SPS genes. Using the Monsanto maize EST and genomic sequence databases, we have identified five full length and two partial SPS sequences, bringing the total number of presently known maize SPS genes to at least seven. Phylogenetic analysis of all known SPS sequences revealed several putative evolutionary branches of SPS. We have classified SPS genes into three major groups in higher plants, all with distinct features from the known microbial SPS genes. Furthermore, this analysis suggests evolutionary divergence of monocotyledonous (monocot) and dicotyledonous (dicot) SPS sequences. The evidence suggests that several gene duplication events occurred at various points during evolution, both before and after the monocot/dicot split. It appears that at least one of the major forms of SPS genes may have evolved after the divergence of monocots and dicots. In addition, several more recent gene duplication events may have occurred after maize/rice speciation, giving rise to additional SPS genes in maize. Some of the variants lack one or more of the presently known regulatory sites, implying that this evolutionary divergence may have given rise to enzymes with functional differences. We present evidence from transcript distribution studies using cDNA libraries as well as transcriptional profiling experiments and propose that specific SPS genes have diverse patterns of expression that are sometimes responsive to environmental signals. Our data suggests that higher plant SPS isozymes differ with respect to their patterns of expression and regulation and that our proposed phylogenetic classification reflects specific functional categories for higher plant SPS isozymes.

The carboxyterminal processing protease of D1 protein: expression, purification and enzymology of the recombinant and native spinach proteins.

The carboxyterminal processing protease of D1 protein (CtpA) is predicted to be an excellent target for a general broad-spectrum herbicide. The gene for spinach CtpA has been expressed in Escherichia coli. The expressed protein that was found mainly in inclusion bodies has been purified and refolded on a nickel-chelate column. Active recombinant CtpA was recovered. Two assays for CtpA activity were developed, a medium-throughput HPLC assay using a fluorescent substrate and a high-throughput assay based on fluorescence polarization capable of application in a high-throughput 96-well plate format. This high-throughput assay was developed to screen chemistry for CtpA inhibitors. Native spinach CtpA was partially purified and the native and recombinant enzymes were compared kinetically for their K(m) and V(max) values using different peptide substrates. Native CtpA partially purified from spinach was shown to have similar kinetic properties to recombinant CtpA. Antibodies developed against the recombinant protein were used to estimate the in planta abundance of the native enzyme in spinach. Since only a small proportion of the recombinant protein is refolded during isolation and it appears that only a small proportion of this enzyme is active, size-exclusion chromatography and light scattering experiments were performed on rCtpA in order to gain insight into its structure and the reasons why most of the protein is not active. The use of rCtpA to screen for herbicidal compounds and the more general question of how good a herbicide target the enzyme is are discussed.

A kinetic comparison of asparagine synthetase isozymes from higher plants.

Four previously identified maize asparagine synthetase (AsnS) genes and a soy AsnS gene have been cloned and expressed in Escherichia coli. The enzymes have been purified and kinetically characterized. The plant AsnS proteins were expressed mainly in the inclusion bodies although small amounts of one form (ZmAsnS2) were recovered in the soluble fraction. In order to measure the kinetic properties of these enzymes a sensitive assay based on the detection of Asn by HPLC has been developed. In addition a method to refold the recombinant plant AsnS to produce active enzyme has been developed. The plant AsnS enzymes are kinetically distinct with substantial differences in K(m) (Gln) and V(max) values when compared to each other. These differences may be important factors for transgenic studies using AsnS genes for crop improvement.