Acetyltransfer in natural product biosynthesis--functional cloning and molecular analysis of vinorine synthase

Vinorine synthase (EC 2.3.1.160) catalyses the acetyl-CoA- or CoA-dependent reversible formation of the alkaloids vinorine (or 11-methoxy-vinorine) and 16-epi-vellosimine (or gardneral). The forward reaction leads to vinorine, which is a direct biosynthetic precursor along the complex pathway to the monoterpenoid indole alkaloid ajmaline, an antiarrhythmic drug from the Indian medicinal plant Rauvolfia serpentina. Based on partial peptide sequences a cDNA clone was isolated and functionally expressed in Escherichia coli. The Km values of the native enzyme for gardneral and acetyl-CoA were determined to be 7.5 and 57 microM. The amino acid sequence of vinorine synthase has highest level of identity (28-31%) to that of Papaver salutaridinol acetyltransferase, Fragaria alcohol acyltransferase, and Catharanthus deacetylvindoline acetyltransferase involved in morphine, flavor, and vindoline biosynthesis, respectively. Vinorine synthase is a novel member of the BAHD superfamily of acyltransferases. Site-directed mutagenesis of 13 amino acid residues provided clear evidence that both, His160 and Asp164 of the consensus sequence HxxxD belong to the catalytic center. The mutations also showed that an amino acid triad is not characteristic of vinorine synthase. The experiments demonstrated the importance of the conserved motif SxL/I/VD near the N-terminus and the consensus sequence DFGWG near the C-terminal.     

Carbon metabolite feedback regulation of leaf photosynthesis and development

Photosynthesis is regulated as a two-way process. Light regulates the expression of genes for photosynthesis and the activity of the gene products (feedforward control). Rate of end-product use down-stream of the Calvin cycle, determined largely by nutrition and temperature, also affects photosynthetic activity and photosynthetic gene expression (feedback control). Whereas feedforward control ensures efficient light use, feedback mechanisms ensure that carbon flow is balanced through the pathways that produce and consume carbon, so that inorganic phosphate is recycled and nitrogen is distributed optimally to different processes to ensure growth and survival. Actual mechanisms are sketchy and complex, but carbon to nitrogen balance rather than carbon status per se is central to understanding carbon metabolite feedback control of photosynthesis. In addition to determining the activity of the metabolic machinery, carbon metabolite feedback mechanisms also regulate photosynthesis at the leaf level through the regulation of leaf development. This review summarizes the current sketchy, but growing, knowledge of the mechanisms through which carbon metabolite feedback mechanisms regulate leaf photosynthesis.     

Structural and biochemical evidence for an autoinhibitory role for tyrosine 984 in the juxtamembrane region of the insulin receptor

Tyrosine 984 in the juxtamembrane region of the insulin receptor, between the transmembrane helix and the cytoplasmic tyrosine kinase domain, is conserved among all insulin receptor-like proteins from hydra to humans. Crystallographic studies of the tyrosine kinase domain and proximal juxtamembrane region reveal that Tyr-984 interacts with several other conserved residues in the N-terminal lobe of the kinase domain, stabilizing a catalytically nonproductive position of alpha-helix C. Steady-state kinetics measurements on the soluble kinase domain demonstrate that replacement of Tyr-984 with phenylalanine results in a 4-fold increase in kcat in the unphosphorylated (basal state) enzyme. Moreover, mutation of Tyr-984 in the full-length insulin receptor results in significantly elevated receptor phosphorylation levels in cells, both in the absence of insulin and following insulin stimulation. These data demonstrate that Tyr-984 plays an important structural role in maintaining the quiescent, basal state of the insulin receptor. In addition, the structural studies suggest a possible target site for small molecule activators of the insulin receptor, with potential use in the treatment of noninsulin-dependent diabetes mellitus.     

Clamp-loader-helicase interaction in Bacillus. Leucine 381 is critical for pentamerization and helicase binding of the Bacillus tau protein

Recently, we revealed the architecture of the clamp-loader-helicase (tau-DnaB) complex in Bacillus by atomic force microscopy imaging and constructed a structural model, whereby a pentameric clamp-loader interacts with the hexameric helicase. Crucial to this model is the assumption that the clamp-loader forms a pentamer in the absence of other components of the clamp-loader complex such as deltadelta'. Here, we show that the Bacillus subtilis tau protein, even in the absence of deltadelta', interacts as a pentamer with the hexameric DnaB and that the L381 of tau is critical for the integrity of the tau oligomer and interaction with DnaB. The effects of the L381A mutation were confirmed by gel filtration, ultracentrifugation, circular dichroism, cross-linking studies, and genetic replacement of the dnaX gene with a mutant L381A dnaX gene in vivo. The L381A protein is able to support growth in vivo only when expressed in high quantities. Finally, despite the fact that a mutation at P465 has been reported to result in a thermosensitive gene in vivo, a P465L mutant protein interacts with DnaB in vitro suggesting that this defect is not a result of a defective tau-DnaB interaction.     

Backbone dynamics of green fluorescent protein and the effect of histidine 148 substitution

Green fluorescent protein (GFP) and its mutants have become valuable tools in molecular biology. GFP has been regarded as a very stable and rigid protein with the beta-barrel shielding the chromophore from the solvent. Here, we report the 15N nuclear magnetic resonance (NMR) studies on the green fluorescent protein (GFPuv) and its mutant His148Gly. 15N NMR relaxation studies of GFPuv show that most of the beta-barrel of GFP is rigid on the picosecond to nanosecond time scale. For several regions, including the first alpha-helix and beta-sheets 3, 7, 8, and 10, increased hydrogen-deuterium exchange rates suggest a substantial conformational flexibility on the microsecond to millisecond time scales. Mutation of residue 148 located in beta-sheet 7 is known to have a strong impact on the fluorescence properties of GFPs. UV absorption and fluorescence spectra in combination with 1H-15N NMR spectra indicate that the His148Gly mutation not only reduces the absorption of the anionic chromophore state but also affects the conformational stability, leading to the appearance of doubled backbone amide resonances for a number of residues. This suggests the presence of two conformations in slow exchange on the NMR time scale in this mutant.     

Dimer-tetramer transition between solution and crystalline states of streptavidin and avidin mutants

The biotin-binding tetrameric proteins, streptavidin from Streptomyces avidinii and chicken egg white avidin, are excellent models for the study of subunit-subunit interactions of a multimeric protein. Efforts are thus being made to prepare mutated forms of streptavidin and avidin, which would form monomers or dimers, in order to examine their effect on quaternary structure and assembly. In the present communication, we compared the crystal structures of binding site W-->K mutations in streptavidin and avidin. In solution, both mutant proteins are known to form dimers, but upon crystallization, both formed tetramers with the same parameters as the native proteins. All of the intersubunit bonds were conserved, except for the hydrophobic interaction between biotin and the tryptophan that was replaced by lysine. In the crystal structure, the binding site of the mutated apo-avidin contains 3 molecules of structured water instead of the 5 contained in the native protein. The lysine side chain extends in a direction opposite that of the native tryptophan, the void being partially filled by an adjacent lysine residue. Nevertheless, the binding-site conformation observed for the mutant tetramer is an artificial consequence of crystal packing that would not be maintained in the solution-phase dimer. It appears that the dimer-tetramer transition may be concentration dependent, and the interaction among subunits obeys the law of mass action.     

Integration site for Streptomyces phage phiBT1 and development of site-specific integrating vectors

Despite extensive similarities between the genomes of the Streptomyces temperate phages phiC31 and phiBT1, the attP-int loci are poorly conserved. Here we demonstrate that phiBT1 integrates into a different attachment site than phiC31. phiBT1 attB lies within SCO4848 encoding a 79-amino-acid putative integral membrane protein. Integration vectors based on phiBT1 integrase were shown to have a broad host range and are fully compatible with those based on the phiC31 attP-int locus.     

PYK2 expression and phosphorylation increases in pressure overload-induced left ventricular hypertrophy

Proline-rich tyrosine kinase 2 (PYK2) is a member of the focal adhesion kinase (FAK) family of nonreceptor protein tyrosine kinases. PYK2 has been implicated in linking G protein-coupled receptors to activation of mitogen-activated protein kinase cascades and cellular growth in a variety of cell types. To determine whether PYK2 expression and phosphorylation is altered in left ventricular (LV) myocardium undergoing LV hypertrophy (LVH) and heart failure in vivo, suprarenal abdominal aortic coarctation was performed in 160-g male Sprague-Dawley rats. Immunohistochemistry and Western blotting were performed on LV tissue 1, 8, and 24 wk after aortic banding. Aortic banding produced sustained hypertension and gradually developing LVH. PYK2 levels were increased 1.8 +/- 0.2-, 2.7 +/- 0.6-, and 2.0 +/- 0.2-fold in 1-, 8-, and 24-wk banded animals compared with their respective sham-operated controls. The increase in PYK2 expression was paralleled by an increase in PYK2 phosphorylation, both of which preceded the development of LVH. Immunohistochemistry revealed that enhanced PYK2 expression occurred predominantly in the cardiomyocyte population. Furthermore, there was a high degree of correlation (R = 0.75; P < 0.001) between the level of PYK2 and the degree of LVH in 24-wk sham and banded animals. In contrast, FAK levels and FAK phosphorylation were not increased before the development of LVH. However, there was a high degree of correlation (R = 0.68; P < 0.001) between the level of FAK and the degree of LVH in 24-wk sham and banded rats. There was also a significant increase in the ratio of phosphospecific anti-FAK to FAK at this time point. These data are consistent with a role for PYK2 in the induction of pressure overload-induced cardiomyocyte hypertrophy, and suggest that PYK2 and FAK have distinctly different roles in LVH progression.     

Regulation of calcium/calmodulin-dependent protein kinase II docking to N-methyl-D-aspartate receptors by calcium/calmodulin and alpha-actinin

Ca(2+) influx through the N-methyl-d-aspartate (NMDA)-type glutamate receptor leads to activation and postsynaptic accumulation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and ultimately to long term potentiation, which is thought to be the physiological correlate of learning and memory. The NMDA receptor also serves as a CaMKII docking site in dendritic spines with high affinity binding sites located on its NR1 and NR2B subunits. We demonstrate that high affinity binding of CaMKII to NR1 requires autophosphorylation of Thr(286). This autophosphorylation reduces the off rate to a level (t(12) = approximately 23 min) that is similar to that observed for dissociation of the T286D mutant CaMKII (t(12) = approximately 30 min) from spines after its glutamate-induced accumulation (Shen, K., Teruel, M. N., Connor, J. H., Shenolikar, S., and Meyer, T. (2000) Nat. Neurosci. 3, 881-886). CaMKII as well as the previously identified NR1 binding partners calmodulin and alpha-actinin bind to the short C-terminal portion of the C0 region of NR1. Like Ca(2+)/calmodulin, autophosphorylated CaMKII competes with alpha-actinin-2 for binding to NR1. We conclude that the NR1 C0 region is a key site for recruiting CaMKII to the postsynaptic site, where it may act in concert with calmodulin to modulate the stimulatory role of alpha-actinin interaction with the NMDA receptor.