The class I terpenoid cyclase epi-isozizaene synthase (EIZS) utilizes the universal achiral isoprenoid substrate, farnesyl diphosphate, to generate epi-isozizaene as the predominant sesquiterpene cyclization product and at least five minor sesquiterpene products, making EIZS an ideal platform for the exploration of fidelity and promiscuity in a terpenoid cyclization reaction. The hydrophobic active site contour of EIZS serves as a template that enforces a single substrate conformation, and chaperones subsequently formed carbocation intermediates through a well-defined mechanistic sequence. Here, we have used the crystal structure of EIZS as a guide to systematically remold the hydrophobic active site contour in a library of 26 site-specific mutants. Remolded cyclization templates reprogram the reaction cascade not only by reproportioning products generated by the wild-type enzyme but also by generating completely new products of diverse structure. Specifically, we have tripled the overall number of characterized products generated by EIZS. Moreover, we have converted EIZS into six different sesquiterpene synthases: F96A EIZS is an (E)-β-farnesene synthase, F96W EIZS is a zizaene synthase, F95H EIZS is a β-curcumene synthase, F95M EIZS is a β-acoradiene synthase, F198L EIZS is a β-cedrene synthase, and F96V EIZS and W203F EIZS are (Z)-γ-bisabolene synthases. Active site aromatic residues appear to be hot spots for reprogramming the cyclization cascade by manipulating the stability and conformation of critical carbocation intermediates. A majority of mutant enzymes exhibit only relatively modest 2–100-fold losses of catalytic activity, suggesting that residues responsible for triggering substrate ionization readily tolerate mutations deeper in the active site cavity.
Potassium (K ϩ ) channels are essential to neuronal signaling and survival. Here we show that these proteins are targets of reactive oxygen species in mammalian brain and that their oxidation contributes to neuropathy. Thus, the KCNB1 (Kv2.1) channel, which is abundantly expressed in cortex and hippocampus, formed oligomers upon exposure to oxidizing agents. These oligomers were ϳ10-fold more abundant in the brain of old than young mice. Oxidant-induced oligomerization of wild-type KCNB1 enhanced apoptosis in neuronal cells subject to oxidative insults. Consequently, a KCNB1 variant resistant to oxidation, obtained by mutating a conserved cysteine to alanine, (C73A), was neuroprotective. The fact that oxidation of KCNB1 is toxic, argues that this mechanism may contribute to neuropathy in conditions characterized by high levels of oxidative stress, such as Alzheimer's disease (AD). Accordingly, oxidation of KCNB1 channels was exacerbated in the brain of a triple transgenic mouse model of AD (3xTg-AD). The C73A variant protected neuronal cells from apoptosis induced by incubation with -amyloid peptide (A 1-42 ). In an invertebrate model (Caenorhabditis elegans) that mimics aspects of AD, a C73A-KCNB1 homolog (C113S-KVS-1) protected specific neurons from apoptotic death induced by ectopic expression of human A 1-42 . Together, these data underscore a novel mechanism of toxicity in neurodegenerative disease. IntroductionPotassium (K ϩ ) channels are a diverse and ubiquitous family of ion-channels that operate in nonexcitable and excitable cells. K ϩ channels are essential for the function of the nervous system where they modulate the shape and frequency of action potentials and consequently, neurotransmitter release. As such, conditions that result in the impairment of K ϩ channels have the potential to cause neuronal dysfunction by affecting the excitability of the cell, which is intrinsically dependent on these proteins. For example, mutations in KCNQ2, KCNQ3, KCNMA1, KCNA1 and KCNC3 genes which lead to the synthesis of mutants with defective properties have been identified in patients affected by epilepsy, ataxia and episodic ataxia type 1 (for review, see Kullmann, 2002). In addition to genetic predisposition, acquired impairment of K ϩ channel's function can lead to neurological diseases, such as, acquired neuromyotonia, limbic encephalitis, and Morvan's syndrome (for review, see Vernino, 2007).Oxygen metabolism leads to the synthesis of reactive and thus potentially toxic molecules known as reactive oxygen species (ROS). The increase in unbuffered ROS levels in a cell, a phenomenon commonly referred to as oxidative stress, is thought to cause significant cellular damage and to contribute to cellular aging (Harman, 1956). Moreover, oxidative stress is elevated in neuropathies such as Alzheimer's disease (AD) (for review, see Lin and Beal, 2006). Oxidative damage occurs early in AD, before the onset of significant plaque formation (Nunomura et al., 2001;Praticò et al., 2001;Reddy et al., 2004), and consequent...
Six amphiphilic heptapeptides with the structure (C 18 H 37 ) 2 NCO-CH 2 OCH 2 CO-(Gly) 3 -Pro-(Gly) n -(Glx)-(Gly) m -O(CH 2 ) 6 CH 3 , in which Glx represents glutamic acid or its benzyl ester and n+m=2, have been studied. In addition, the glutamate residue in the GGGPGGE sequence was esterified by fluorescent 1-pyrenemethanol. These compounds insert into phospholipid bilayers and form anionconducting pores. Hill plots based on carboxyfluorescein release indicate that the pores are at least dimeric. Studies that involved ion-selective electrode techniques showed that transport of chloride varied with the position of glutamate within the peptide chain and whether glutamic acid was present as the free acid or its benzyl ester. Chloride transport activity was significantly higher for the glutamate esters than for free carboxylates irrespective of the glutamate position. Activity was highest when the glutamate residue in ∼(Gly) 3 -Pro-(Xxx) 3 ∼ was closest to the C terminus of the peptide. A fluorescent pyrene residue was introduced to probe the aggregation state of the amphiphile. The selectivity of the pore for Cl -over K + was maintained even when the carboxylate anion was present within it. Complexation of Cl -by the ionophoric peptides was confirmed by negative ion mass spectrometry. Planar bilayer voltage clamp experiments confirmed that pores with more than one conductance state may form in these dynamic, self-assembled pores.
Hexaploid wheat (Triticum aestivum L.) is an important food crop but it is vulnerable to heat. The heat-responsive proteome of wheat remains to be fully elucidated because of previous technical and genomic limitations, and this has hindered our understanding of the mechanisms of wheat heat adaptation and advances in improving thermotolerance. Here, flag leaves of wheat during grain filling stage were subjected to high daytime temperature stress, and 258 heat-responsive proteins (HRPs) were identified with iTRAQ analysis. Enrichment analysis revealed that chlorophyll synthesis, carbon fixation, protein turnover, and redox regulation were the most remarkable heat-responsive processes. The HRPs involved in chlorophyll synthesis and carbon fixation were significantly decreased, together with severe membrane damage, demonstrating the specific effects of heat on photosynthesis of wheat leaves. In addition, the decrease in chlorophyll content may result from the decrease in HRPs involved in chlorophyll precursor synthesis. Further analysis showed that the accumulated effect of heat stress played a critical role in photosynthesis reduction, suggested that improvement in heat tolerance of photosynthesis, and extending heat tolerant period would be major research targets. The significantly accumulation of GSTs and Trxs in response to heat suggested their important roles in redox regulation, and they could be the promising candidates for improving wheat thermotolerance. In summary, our results provide new insight into wheat heat adaption and provide new perspectives on thermotolerance improvement.
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