Arginyl residues and anion binding sites in proteins

Summary The functions of a number of amino acid residues in proteins have been studied by chemical modification techniques and much useful information has been obtained. Methods using dicarbonyl compounds for the modification of arginine residues are the most recent to have been developed. Since their introduction about 10 years ago, they have led to the identification of a large number of enzymes and other proteins that contain arginine residues critical to biological function. These reagents are discussed in terms of their chemical reactivity and mechanisms of action and in relation to the unique chemical properties of the guanidinium group. Butanedione, phenylglyoxal and cyclohexanedione are the most commonly employed arginyl reagents, and their relative advantages are examined. A survey of the functional role of arginine residues in enzymes and other proteins is presented in which nearly 100 examples are cited. The prediction that arginine residues would be found to serve a general role as anionic binding sites in protein has obviously been validated. The genetic and physiological implications of the selection of arginine for this important function are discussed.This work was supported by Grant-in-Aid GM-15003 from the National Institutes of Health.     

Efficient,environmentally-friendly and specific valorization of lignin: promising role of non-radical lignolytic enzymes

Lignin is the second most abundant bio-resource in nature. It is increasingly important to convert lignin into high value-added chemicals to accelerate the development of the lignocellulose biorefinery. Over the past several decades, physical and chemical methods have been widely explored to degrade lignin and convert it into valuable chemicals. Unfortunately, these developments have lagged because of several difficulties, of which high energy consumption and non-specific cleavage of chemical bonds in lignin remain the greatest challenges. A large number of enzymes have been discovered for lignin degradation and these are classified as radical lignolytic enzymes and non-radical lignolytic enzymes. Radical lignolytic enzymes, including laccases, lignin peroxidases, manganese peroxidases and versatile peroxidases, are radical-based bio-catalysts, which degrade lignins through non-specific cleavage of chemical bonds but can also catalyze the radical-based re-polymerization of lignin fragments. In contrast, non-radical lignolytic enzymes selectively cleave chemical bonds in lignin and lignin model compounds and, thus, show promise for use in the preparation of high value-added chemicals. In this mini-review, recent developments on non-radical lignolytic enzymes are discussed. These include recently discovered non-radical lignolytic enzymes, their metabolic pathways for lignin conversion, their recent application in the lignin biorefinery, and the combination of bio-catalysts with physical/chemical methods for industrial development of the lignin refinery.     

Sequence homology analysis of a heterogenous protein population by chemical and enzymic digestion using a two-dimensional sodium dodecyl sulfate-polyacrylamide gel system

A procedure for examining possible sequence homology between two or more proteins in a heterogenous protein mixture using a two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis system is described. Three different chemical reagents (cyanogen bromide, hydroxylamine, and acetic acid) and three enzymes (α-chymotrypsin, trypsin, and Staphylococcus aureus protease) have been used as the cleavage reagents for the peptide mapping studies. Potential application of this technique in conjunction with radioactive labeling and immunological studies was also demonstrated.     

Peptides as modulators of enzymes and regulatory proteins

There is currently great interest in the development of methods to modulate the function of diverse classes of target proteins with chemicals (agonists or antagonists). These would be valuable reagents for biomedical research and some might serve as potential drug leads. Traditionally, most chemicals that modulate protein function have been enzyme inhibitors isolated in functional screens specific for the enzyme of interest. However, recent efforts from many laboratories have suggested that relatively simple binding assays may provide a more convenient and general route to chemical modulators. We review here this work with a particular emphasis on peptide modulators.     

生物转化-从全细胞催化到代谢工程

与传统的化学合成方法相比,利用生物的手段转化生产活性化合物及其衍生物无疑具有更大的吸引力。随着用于生物转化微生物种类的增多,生物转化的应用领域不断得到扩大。生物转化的发展经历了野生型全细胞催化,基因工程微生物全细胞反应,以及利用系统分析和代谢工程进行全局性调控等几个阶段。以下对这一发展趋势及相关研究的最新进展作一简要综述。     

Accessibility and cross-linking of native neurofilaments to chemical reagents

Native neurofilaments were submitted to cross-linking reactions with bifunctional reagents (DMA, DMS and DSS) and to chemical reactions with sterically bulky reagents such as EEDQ and DTAF , as well as a glutaraldehyde-activated gel. The 160K and 70K neurofilament proteins reacted slightly more than the 210K neurofilament protein with DMS and DSS. The accessibility of the three neurofilaments to the other chemical reagents was identical. These results were unexpected since neurofilament antibodies seem to react preferentially with 210K protein which is at the periphery of the filament, whereas the 70K protein, which is the backbone of the filament, is probably buried inside the filament. In the same way, it has been shown that the side of the 210K proteins are probably able to cross link the neurofilaments with non covalent and covalent bridges. Using different cross link reagents, we did not observe a characteristic reactivity of the 210K protein towards the different chemicals. We conclude that the three neurofilament proteins are equally exposed to the different sterically bulky reagent and that part of the polypeptide chain of the 70K and the 160K proteins are located at the outside of the filament.     

Laboratory evolution of catabolic enzymes and pathways

The laboratory evolution of environmentally relevant enzymes and proteins has resulted in the generation of optimized and stabilized enzymes, as well as enzymes with activity against new substrates. Numerous methods, including random mutagenesis, site-directed mutagenesis and DNA shuffling, have been widely used to generate variants of existing enzymes. These evolved catabolic enzymes have application for improving biodegradation pathways, generating engineered pathways for the degradation of particularly recalcitrant compounds, and for the development of biocatalytic processes to produce useful compounds. Regulatory proteins associated with catabolic pathways have been utilized to generate biosensors for the detection of bioavailable concentrations of environmentally relevant chemicals.     

Biochemical strategies for the detection and detoxification of toxic chemicals in the environment

Addressing the problems related to the widespread presence of an increasing number of chemicals released into the environment by human activities represents one of the most important challenges of this century. In the last few years, to replace the high cost, in terms of time and money, of conventional technologies, the scientific community has directed considerable research towards the development both of new detection systems for the measurement of the contamination levels of chemicals in people's body fluids and tissue, as well as in the environment, and of new remediation strategies for the removal of such chemicals from the environment, as a means of the prevention of human diseases. New emerging biosensors for the analysis of environmental chemicals have been proposed, including VHH antibodies, that combine the antibody performance with the affinity for small molecules, genetically engineered microorganisms, aptamers and new highly stable enzymes. However, the advances in the field of chemicals monitoring are still far from producing a continuous realtime and on-line system for their detection. Better results have been obtained in the development of strategies which use organisms(microorganisms, plants and animals) or metabolic pathway-based approaches(single enzymes or more complex enzymatic solutions) for the fixation, degradation and detoxification of chemicals in the environment. Systems for enzymatic detoxification and degradation of toxic agents in wastewater from chemical and manufacturing industries, such as ligninolytic enzymes for the treatment of wastewater from the textile industry, have been proposed. Considering the high value of these research studies, in terms of the protection of human health and of the ecosystem, science must play a major role in guiding policy changes in this field.     

In vitro and in vivo extrapolations of genotoxin exposures: consideration of factors which influence dose-response thresholds

The concept of a threshold of activity of a genotoxic agent is primarily based upon considerations of protective mechanisms and multiple cellular targets, which require inactivation before a toxic response is produced. In this paper, we have considered and evaluated the influences of compound metabolism, DNA lesion formation, mutation induction and sequence content, aneuploidy induction and the influence of repair enzymes upon genetic endpoints produced by both DNA reactive chemicals and by those chemicals which modify non-DNA cellular targets. Thresholds of activity have been evaluated by critical analysis of the published literature and original data analysing both the role of sequence context upon point mutation induction and DNA repair mechanisms upon the sensitivity of cultured cells to the induction of aneuploidy. In the case of DNA reactive chemicals, the presence of a threshold of chemical activity will be dependent upon cellular activities such as those of the Phase II enzymes reducing the activity of chemicals before lesion formation takes place and/or those of the DNA repair enzymes which reduce the proportion of DNA lesions which are processed into DNA sequence changes. Under such conditions, a given exposure of a DNA reactive chemical does not produce a linear or semi-linear increase in DNA lesions or in mutation frequency. However, even when these protective mechanisms are overwhelmed by the high exposures of genotoxic chemicals the biological effects of a genotoxin may be influenced by the sequence context of the gene under consideration. Here, we demonstrate that point mutations are detected at relatively higher frequencies in the non-coding introns compared with the coding exons. Many of the base changes detected in the exons do not produce amino acid changes in the proteins coded for by the genes being monitored for mutation induction. Both sequence context and the types of base changes induced may provide a "buffering" effect reducing the biological consequences of mutation induction. Spindle damaging chemicals, such as colcemid and vinblastine, induce aneuploidy by modifying the numbers of spindle fibres which regulate the segregation of chromosomes during mitosis and meiosis. The redundancy of spindle fibres in the dividing mammalian cell leads to the prediction that only chemical exposures which damage most, if not all, of the fibres will lead to the induction of polyploidy and/or aneuploidy. Such predicted thresholds of chemical activity can be observed when both chromosome loss and non-disjunction are measured in wild type cultures. However, we observed a substantial increase in sensitivity to aneugenic chemicals when measurements were made in primary cell cultures derived from xerodoma pigmentosum and trichothiodystrophy patients. Further studies are necessary to evaluate the consequences of the genetic background of tester strains upon the nature of the dose-response curve of aneugenic chemicals.     

Electrochemical synthesis and impedance characterization of nano-patterned biosensor substrate

The nano-porous anodic aluminum oxide has been used as a substrate material for enzymatic biosensor operating in aqueous solutions. Nano-scale porous structure was formed by electrical anodization in an acid solution. By changing anodization conditions, such as electrolyte concentration, temperature, and anodization time, the ordered hexagonal porous structure with well-controlled pore size and depth can be obtained. Nano-porous alumina substrate with adsorbed enzymes was used as an enzyme electrode and pH sensor. The pH changes are driven by the enzymatic reactions, e.g. penicillin G hydrolysis to form penicilloic acid in the presence of penicillinaze. The advantage of physical adsorption used to bound penicillinaze, the model enzyme in this work, to the porous structure, is that usually no reagents are required and only a minimum of "activation" or clean-up steps. Adsorption tends to be less disruptive to enzyme proteins than chemical attachment. Due to the increased active sensor area, the immobilization of enzymes has been enhanced, which in turn improved the electrode's sensitivity. To characterize the interactions of enzymes with nano-porous alumina oxide, electrochemical impedance spectroscopy (EIS) was used.     

Production of bulk chemicals via novel metabolic pathways in microorganisms

Metabolic engineering has been playing important roles in developing high performance microorganisms capable of producing various chemicals and materials from renewable biomass in a sustainable manner. Synthetic and systems biology are also contributing significantly to the creation of novel pathways and the whole cell-wide optimization of metabolic performance, respectively. In order to expand the spectrum of chemicals that can be produced biotechnologically, it is necessary to broaden the metabolic capacities of microorganisms. Expanding the metabolic pathways for biosynthesizing the target chemicals requires not only the enumeration of a series of known enzymes, but also the identification of biochemical gaps whose corresponding enzymes might not actually exist in nature; this issue is the focus of this paper. First, pathway prediction tools, effectively combining reactions that lead to the production of a target chemical, are analyzed in terms of logics representing chemical information, and designing and ranking the proposed metabolic pathways. Then, several approaches for potentially filling in the gaps of the novel metabolic pathway are suggested along with relevant examples, including the use of promiscuous enzymes that flexibly utilize different substrates, design of novel enzymes for non-natural reactions, and exploration of hypothetical proteins. Finally, strain optimization by systems metabolic engineering in the context of novel metabolic pathways constructed is briefly described. It is hoped that this review paper will provide logical ways of efficiently utilizing ‘big’ biological data to design and develop novel metabolic pathways for the production of various bulk chemicals that are currently produced from fossil resources.     

Strategy for Stabilizing Enzymes Part Two: Increasing Enzyme Stability by Selective Chemical Modification

The molecular mechanisms of change in the thermal stability of proteins modified with low molecular weight reagents are discussed. The choice of stabilization mechanisms to be used as a general strategy for increasing enzyme stability by chemical modification is addressed. Hydrophilization of nonpolar surface areas is the most simple and reliable approach to artificial stabilization of enzymes for use in applied biochemistry and biotechnology.     

Quorum quenching and proactive host defense

Both plants and humans have inducible defense mechanisms. This passive defense strategy leaves the host unprotected for a period of time until resistance is activated. Moreover, many bacterial pathogens have evolved cell-cell communication (quorum-sensing) mechanisms to mount population-density-dependent attacks to overwhelm the host's defense responses. Several chemicals and enzymes have been investigated for years for their potential to target the key components of bacterial quorum-sensing systems. These quorum-quenching reagents, which block bacterial cell-cell communications, can disintegrate a bacterial population-density-dependent attack. It has now been shown that a quorum-quenching mechanism can be engineered in plants and might be used as a strategy in controlling bacterial pathogens and to build up a proactive defense barrier.     

Applications of chemical cleavage procedures to the peptide mapping of neurofilament triplet protein bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis

A procedure for examining possible sequence homology in the triplet neurofilament proteins using a sodium dodecyl sulfate-polyacrylamide gel electrophoresis system is described. Five different chemical reagents (cyanogen bromide, BNPS-skatole, hydroxylamine, formic acid, and nitrothiocyanobenzoic acid) have been used for peptide mapping studies. Potential applications of this technique are discussed.     

Cleavable linkers in chemical biology

Interest in cleavable linkers is growing due to the rapid development and expansion of chemical biology. The chemical constrains imposed by the biological conditions cause significant challenges for organic chemists. In this review we will present an overview of the cleavable linkers used in chemical biology classified according to their cleavage conditions by enzymes, nucleophilic/basic reagents, reducing agents, photo-irradiation, electrophilic/acidic reagents, organometallic and metal reagents, oxidizing reagents.     

Study of P450 function using gene knockout and transgenic mice

The xenobiotic-metabolizing P450s have been extensively studied for their ability to metabolize endogenous and exogenous chemicals. The latter include drugs and dietary and environmentally derived toxicants and carcinogens. These enzymes also metabolize endogenous steroids and fatty acids. P450s are thought to be required for efficient removal of most xenobiotics from the body and to be responsible for the hazardous effects of toxicants and carcinogens based on their ability to convert chemicals to electrophilic metabolites that can cause cellular damage and gene mutations. P450 catalytic activities have been extensively studied in vitro and in cell culture, yielding considerable information on their mechanisms of catalysis, substrate specificities, and metabolic products. Targeted gene disruption has been used to determine the roles of P450s in intact animals and their contributions to the mechanisms of toxicity and carcinogenesis. The P450s chosen for study, CYP1A1, CYP1B1, CYP1A2, and CYP2E1, are conserved in mammals and are known to metabolize most toxicants and chemical carcinogens. Mice lacking expression of these enzymes do not differ from wild-type mice, indicating that these P450s are not required for development and physiological homeostasis. However, the P450 null mice have altered responses to the toxic and carcinogenic effects of chemicals as compared with wild-type mice. These studies establish that P450s mediate the adverse effects of drugs and dietary, environmental, and industrial chemicals and serve to validate molecular epidemiology studies that seek to determine links between P450 polymorphisms and susceptibility to chemically associated diseases. More recently, P450 humanized mice have been produced.     

Solid-Phase Cross-Linking (SPCL): a new tool for protein structure studies

A wide range of chemical reagents are available to study the protein-protein interactions or protein structures. After reaction with such chemicals, covalently modified proteins are digested, resulting in shorter peptides that are analyzed by mass spectrometry (MS). Used especially when NMR of X-ray data are lacking, this methodology requires the identification of modified species carrying relevant information, among the unmodified peptides. To overcome the drawbacks of existing methods, we propose a more direct strategy relying on the synthesis of solid-supported cleavable monofunctional reagents and cross-linkers that react with proteins and that selectively release, after protein digestion and washings, the modified peptide fragments ready for MS analysis. Using this Solid-Phase Cross-Linking (SPCL) strategy, only modified sequences are analyzed and consistent data can be easily obtained since the signals of interest are not masked or suppressed by over-represented unmodified materials.     

Glutathione S-transferase in chemotherapy resistance and in carcinogenesis.

Cytosolic glutathione S-transferases are composed of two monomeric subunits. These monomers are the products of different gene families designated alpha, mu, and pi. Dimerization yields either homodimeric or heterodimeric holoenzymes within the same family. The members of this complex group of proteins have been linked to the detoxification of environmental chemicals and carcinogens, and have been shown to be overexpressed in normal and tumor cells following exposure to cytotoxic drugs. They also are overexpressed in carcinogen-induced rat liver preneoplastic nodules in rat liver. In all of these cases, the changes in expression of glutathione S-transferases are paralleled by increased resistance to cytotoxic chemicals. The degree of resistance is related to the substrate specificity of the isozyme. The relationship of the glutathione S-transferase genes to drug resistance has been directly demonstrated by gene transfer studies, where cDNAs encoding the various subunits of glutathione S-transferase have been transfected into a variety of cell types. This review discusses the results of numerous studies that associate resistance to alkylating agents with overexpression of protective detoxifying glutathione S-transferase enzymes.