Genetic and environmental considerations for evaluating crown position of wheat

Summary Crown position affects winter survival of fallsown wheat (Triticum aestivum L.) Direct or indirect selection for crown depth has been little practiced. Reports have suggested that short subcrown internode length was closely related to semidwarf plant height and that semidwarfism was related to poor emergence. This study determined the relationships among crown depth, plant height, and emergence rate index in three wheat populations. The efficiency of evaluating crown placement in the field was examined and additional information was obtained on its genetic control. The F₂-derived F₄ and F₅ lines from the crosses of female parents Daws, Nugaines, and Stephens with male parent Selection 7952 were planted at Central Ferry and Pullman, Washington, respectively. Correlations from each population indicated that crown depth and subcrown internode length were not closely associated with plant height and emergence rate index. Crown depth was a more reliable indicator of crown placement than subcrown internode length. Adjustment of the data for seed depth differences was essential for evaluating subcrown internode length but less important for evaluating crown depth. After adjustment for seed depth, narrow-sense h ² values for subcrown internode length and crown depth were 0.25–0.41. Crown depth and subcrown internode length were inherited as quantitative traits in phenotypes that expressed variable dominance. Modest gains due to selection for crown depth were achieved.Contribution from USDA-ARS and College of Agriculture and Home Economics Research Center, Washington State University, Scientific Paper No. 7795     

The FMN to heme electron transfer in cytochrome P450BM-3. Effect of chemical modification of cysteines engineered at the FMN-heme domain interaction site

The crystal structure of the complex between the heme and FMN-containing domains of Bacillus megaterium cytochrome P450BM-3 (Sevrioukova, I. F., Li, H., Zhang, H., Peterson, J. A., and Poulos, T. L. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 1863-1868) indicates that the proximal side of the heme domain molecule is the docking site for the FMN domain and that the Pro(382)-Gln(387) peptide may provide an electron transfer (ET) path from the FMN to the heme iron. In order to evaluate whether ET complexes formed in solution by the heme and FMN domains are structurally relevant to that seen in the crystal structure, we utilized site-directed mutagenesis to introduce Cys residues at positions 104 and 387, which are sites of close contact between the domains in the crystal structure and at position 372 as a control. Cys residues were modified with a bulky sulfhydryl reagent, 1-dimethylaminonaphthalene-5-sulfonate-L-cystine (dansylcystine (DC)), to prevent the FMN domain from binding at the site seen in the crystal structure. The DC modification of Cys(372) and Cys(387) resulted in a 2-fold decrease in the rates of interdomain ET in the reconstituted system consisting of the separate heme and FMN domains and had no effect on heme reduction in the intact heme/FMN-binding fragment of P450BM-3. DC modification of Cys(104) caused a 10-20-fold decrease in the interdomain ET reaction rate in both the reconstituted system and the intact heme/FMN domain. This indicates that the proximal side of the heme domain molecule represents the FMN domain binding site in both the crystallized and solution complexes, with the area around residue 104 being the most critical for the redox partner docking.     

Exploring the electron transfer properties of neuronal nitric-oxide synthase by reversal of the FMN redox potential

In nitric-oxide synthase (NOS) the FMN can exist as the fully oxidized (ox), the one-electron reduced semiquinone (sq), or the two-electron fully reduced hydroquinone (hq). In NOS and microsomal cytochrome P450 reductase the sq/hq redox potential is lower than that of the ox/sq couple, and hence it is the hq form of FMN that delivers electrons to the heme. Like NOS, cytochrome P450BM3 has the FAD/FMN reductase fused to the C-terminal end of the heme domain, but in P450BM3 the ox/sq and sq/hq redox couples are reversed, so it is the sq that transfers electrons to the heme. This difference is due to an extra Gly residue found in the FMN binding loop in NOS compared with P450BM3. We have deleted residue Gly-810 from the FMN binding loop in neuronal NOS (nNOS) to give Delta G810 so that the shorter binding loop mimics that in cytochrome P450BM3. As expected, the ox/sq redox potential now is lower than the sq/hq couple. Delta G810 exhibits lower NO synthase activity but normal levels of cytochrome c reductase activity. However, unlike the wild-type enzyme, the cytochrome c reductase activity of Delta G810 is insensitive to calmodulin binding. In addition, calmodulin binding to Delta G810 does not result in a large increase in FMN fluorescence as in wild-type nNOS. These results indicate that the FMN domain in Delta G810 is locked in a unique conformation that is no longer sensitive to calmodulin binding and resembles the "on" output state of the calmodulin-bound wild-type nNOS with respect to the cytochrome c reduction activity.     

Soluble guanylate cyclase

Soluble guanylate cyclase (sGC) is a mammalian nitric oxide (NO) sensor. When NO binds to the sGC heme, its GTP cyclase activity markedly increases, thus generating cyclic GMP, which serves to regulate several cell signaling functions. A good deal is known about the kinetics and equilibrium of binding of NO to sGC, leading to a proposed multistep mechanism of sGC activation that involves at least two NO-binding sites. The crystal structure of a member of a recently discovered family of prokaryotic sGC homologues has provided important insights into structure-function relationships within the sGC family of proteins.     

Taura syndrome virus from Belize represents a unique variant

A Taura syndrome virus (TSV) isolate from cultured Penaeus vannamei grown in Belize, Central America was characterized and shown to be a unique isolate. Mortality rates in laboratory infections of specific pathogen-free (SPF) P. vannamei, reactivity of the virus with monoclonal antibody (MAb) 1A1 and phylogenetic analysis demonstrated that the Belize isolate (BLZ02TSV) is a new valiant of TSV. The Hawaiian 1994 TSV isolate (HI94TSV, GenBank AF277675) was used as the reference isolate for these studies. Laboratory infections of SPF P. vannamei with BLZ02TSV demonstrated higher mortalities and earlier onset of mortalities compared to infections with HI94TSV. Shrimp tissues infected with BLZ02TSV reacted with a TSV-specific gene probe by in situ hybridization and were positive by RT-PCR using TSV diagnostic primers, thus indicating that the isolate was TSV. However, Western blot analysis and immunohistochemistry using MAb 1A1 demonstrated that BLZ02TSV did not react with the antibody, suggestive of changes in the VP1 region of the genome that codes for the polypeptide to which MAb 1A1 binds. Phylogenetic analysis of a 1.3 kbp fragment of the TSV VP1 capsid region revealed that BLZ02TSV represents a distinct group among more than 29 isolates of TSV studied thus far. This research demonstrates that BLZ02TSV is a unique isolate of TSV and reiterates a problem related to the use of MAb 1A1 for detection of TSV in clinical specimens.     

Structural characterization and kinetics of nitric-oxide synthase inhibition by novel N5-(iminoalkyl)- and N5-(iminoalkenyl)-ornithines

Isoform-specific nitric-oxide synthase (NOS) inhibitors may prove clinically useful in reducing the pathophysiological effects associated with increased neuronal NOS (nNOS) or inducible NOS (iNOS) activity in a variety of neurological and inflammatory disorders. Analogs of the NOS substrate L-arginine are pharmacologically attractive inhibitors because of their stability, reliable cell uptake, and good selectivity for NOS over other heme proteins. Some inhibitory arginine analogs show significant isoform selectivity although the structural or mechanistic basis of such selectivity is generally poorly understood. In the present studies, we determined by x-ray crystallography the binding interactions between rat nNOS and N5-(1-imino-3-butenyl)-L-ornithine (L-VNIO), a previously identified mechanism-based, irreversible inactivator with moderate nNOS selectivity. We have also synthesized and mechanistically characterized several L-VNIO analogs and find, surprisingly, that even relatively minor structural changes produce inhibitors that are either iNOS-selective or non-selective. Furthermore, derivatives having a methyl group added to the butenyl moiety of L-VNIO and L-VNIO derivatives that are analogs of homoarginine rather than arginine display slow-on, slow-off kinetics rather than irreversible inactivation. These results elucidate some of the structural requirements for isoform-selective inhibition by L-VNIO and its related alkyl- and alkenyl-imino ornithine and lysine derivatives and may provide information useful in the ongoing rational design of isoform-selective inhibitors.     

A novel heme and peroxide-dependent tryptophan-tyrosine cross-link in a mutant of cytochrome c peroxidase

The crystal structure of a cytochrome c peroxidase mutant where the distal catalytic His52 is converted to Tyr reveals that the tyrosine side-chain forms a covalent bond with the indole ring nitrogen atom of Trp51. We hypothesize that this novel bond results from peroxide activation by the heme iron followed by oxidation of Trp51 and Tyr52. This hypothesis has been tested by incorporation of a redox-inactive Zn-protoporphyrin into the protein, and the resulting crystal structure shows the absence of a Trp51-Tyr52 cross-link. Instead, the Tyr52 side-chain orients away from the heme active-site pocket, which requires a substantial rearrangement of residues 72-80 and 134-144. Additional experiments where heme-containing crystals of the mutant were treated with peroxide support our hypothesis that this novel Trp-Tyr cross-link is a peroxide-dependent process mediated by the heme iron.     

Crystal structures of the ferric,ferrous, and ferrous-NO forms of the Asp140Ala mutant of human heme oxygenase-1: catalytic implications

Site-directed mutagenesis studies have shown that Asp140 in both human and rat heme oxygenase-1 is critical for enzyme activity. Here, we report the D140A mutant crystal structure in the Fe(III) and Fe(II) redox states as well as the Fe(II)-NO complex as a model for the Fe(II)-oxy complex. These structures are compared to the corresponding wild-type structures. The mutant and wild-type structures are very similar, except for the distal heme pocket solvent structure. In the Fe(III) D140A mutant one water molecule takes the place of the missing Asp140 carboxylate side-chain and a second water molecule, novel to the mutant, binds in the distal pocket. Upon reduction to the Fe(II) state, the distal helix running along one face of the heme moves closer to the heme in both the wild-type and mutant structures thus tightening the active site. NO binds to both the wild-type and mutant in a bent conformation that orients the NO O atom toward the alpha-meso heme carbon atom. A network of water molecules provides a H-bonded network to the NO ligand, suggesting a possible proton shuttle pathway required to activate dioxygen for catalysis. In the wild-type structure, Asp140 exhibits two conformations, suggesting a dynamic role for Asp140 in shuttling protons from bulk solvent via the water network to the iron-linked oxy complex. On the basis of these structures, we consider why the D140A mutant is inactive as a heme oxygenase but active as a peroxidase.     

Mesopone cytochrome c peroxidase: functional model of heme oxygenated oxidases

The effect of heme ring oxygenation on enzyme structure and function has been examined in a reconstituted cytochrome c peroxidase. Oxochlorin derivatives were formed by OsO(4) treatment of mesoporphyrin followed by acid-catalyzed pinacol rearrangement. The northern oxochlorin isomers were isolated by chromatography, and the regio-isomers assignments determined by 2D COSY and NOE 1H NMR. The major isomer, 4-mesoporphyrinone (Mp), was metallated with FeCl(2) and reconstituted into cytochrome c peroxidase (CcP) forming a hybrid green protein, MpCcP. The heme-altered enzyme has 99% wild-type peroxidase activity with cytochrome c. EPR spectroscopy of MpCcP intermediate compound I verifies the formation of the Trp(191) radical similar to wild-type CcP in the reaction cycle. Peroxidase activity with small molecules is varied: guaiacol turnover increases approximately five-fold while that with ferrocyanide is approximately 85% of native. The electron-withdrawing oxo-substitutents on the cofactor cause a approximately 60-mV increase in Fe(III)/Fe(II) reduction potential. The present investigation represents the first structural characterization of an oxochlorin protein with X-ray intensity data collected to 1.70 A. Although a mixture of R- and S-mesopone isomers of the FeMP cofactor was used during heme incorporation into the apo-protein, only the S-isomer is found in the crystallized protein.