Enzyme-catalyzed oligosaccharide synthesis.

Cell-surface carbohydrates and their conjugates are involved in many types of molecular recognition. This review describes recent developments in enzyme-catalyzed oligosaccharide synthesis, with particular focus on glycosyltransferase and glycosidase reactions. With the increasing availability of glycosyltransferases via recombinant DNA technology, glycosyltransferase-catalyzed glycosylation with in situ regeneration of sugar nucleotides appears to be the most effective method for large-scale stereocontrolled oligosaccharide synthesis.     

Enzyme-catalyzed synthesis of carbohydrates

Several new enzymes of utility in the synthesis of carbohydrates have been reported during the past year. Additionally, the utility of several well studied enzymes has been expanded. Pyruvate aldolases, aldolase abzymes and both wild-type and mutated glycosidases have found increasing acceptance in the community. Preliminary reports suggest that thermophilic enzymes may possess significant advantages compared to their mesophilic counterparts for carbohydrate synthesis.     

Synthesis and photochemical protein crosslinking studies of hydrophilic naphthalimides

A mixture of 4-alkylamino-1,8-naphthalimides has previously been reported to exhibit potential utility as a photochemical tissue-bonding reagent. In order to determine which constituents of the mixture were responsible for the observed tissue bonding and to facilitate study of the mechanism, we have synthesized each of the primary constituents of the mixture. Each naphthalimide synthesized has been demonstrated to photochemically crosslink proteins.     

A water-soluble, monitorable peptide and protein crosslinking agent

A novel, freely water-soluble, heterobifunctional crosslinking reagent, N-maleimido-6-aminocaproyl ester of 1-hydroxy-2-nitro-4-benzenesulfonic acid (mal-sac-HNSA), was synthesized and used for conjugation of sulfhydryl (cysteine)-containing peptides to carrier proteins. Reaction with amino groups releases the dianion phenolate, HNSA, which allows convenient spectrophotometric quantitation of the reaction in progress. Since mal-sac-HNSA is completely water soluble, its concentration can be adjusted to maximize the rate of amine reaction and to minimize hydrolysis. Conjugates of peptides to appropriate carriers have elicited peptide-specific antibody and did not elicit detectable antibody specific to the crosslink.     

Genetically encoded protein labeling and crosslinking in living Pseudomonas aeruginosa

Pseudomonas aeruginosa (PA) is a major human pathogen for hospital-acquired infections. We report the genetic code expansion of this opportunistic pathogen by using the pyrrolysyl-tRNA synthetase-tRNA system, which enabled the genetic and site-specific incorporation of unnatural amino acids bearing bioorthogonal handles or photo-affinity groups into proteins in PA. This strategy allowed us to conduct bioorthogonal labeling and imaging of flagella, as well as site-specific photo-affinity capturing of interactions between a Type III secretion effector and its chaperone inside living bacteria.     

Complete exchange of phosphatidylcholine from intact erythrocytes after protein crosslinking

Treatment of human erythrocytes with tetrathionate or diamide resulted in extensive crosslinking of membraneous and cytoskeletal proteins. Such treatment was followed by an incubation with phosphatidylcholine specific exchange protein to investigate the rate and extent of exchange of phosphatidylcholine between the erythrocytes and ¹⁴C-labeled phosphatidylcholine containing microsomal membranes or vesicles. Exchange profiles showed that the exchange of phosphatidylcholine is facilitated in treated cells when compared to control erythrocytes and, more importantly, that all of the phosphatidylcholine is exchangeable after protein crosslinking whereas in control cells only the phosphatidylcholine pool located in the outer layer of the membrane is exchangeable. These observations demonstrate that crosslinking of cytoskeletal and membraneous proteins enhances the rate of transbilayer movement of phosphatidylcholine considerably.     

天然多羟基化合物在酶催化下的选择性酯化

综述了近十几年来几种天然多羟基化合物包括皂草苷类、黄酮苷类、生物碱类、甾醇类、倍半萜(烯)化合物、熊果苷和槐糖脂在脂肪酶和枯草杆菌蛋白酶催化下的选择性酯化。     

Dramatic modulation of electron transfer in protein complexes by crosslinking.

The transfer of electrons between proteins is an essential step in biological energy production. Two protein redox partners are often artificially crosslinked to investigate the poorly understood mechanism by which they interact. To better understand the effect of crosslinking on electron transfer rates, we have constructed dimers of azurin by crosslinking the monomers. The measured electron exchange rates, combined with crystal structures of the dimers, demonstrate that the length of the linker can have a dramatic effect on the structure of the dimer and the electron transfer rate. The presence of ordered water molecules in the protein-protein interface may considerably influence the electronic coupling between redox centers.     

AtFim1 is an actin filament crosslinking protein from Arabidopsis thaliana

ATFIM1 is a widely expressed gene in Arabidopsis thaliana that encodes a putative actin filament-crosslinking protein, AtFim1, belonging to the fimbrin/plastin class of actin-binding proteins. In this report we have used bacterially expressed AtFim1 and actin isolated from Zea mays pollen to demonstrate that AtFim1 functions as an actin filament-crosslinking protein. AtFim1 binds pollen actin filaments (F-actin) in a calcium-independent manner, with an average dissociation constant (Kd) of 0.55+/-0.21 microM and with a stoichiometry at saturation of 1:4 (mol AtFim1 : mol actin monomer). AtFim1 also crosslinks pollen F-actin by a calcium-independent mechanism, in contrast to crosslinking of plant actin by human T-plastin, a known calcium-sensitive actin-crosslinking protein. When micro-injected at high concentration into living Tradescantia virginiana stamen hair cells, AtFim1 caused cessation of both cytoplasmic streaming and transvacuolar strand dynamics within 2-4 min. Using the 'nuclear displacement assay' as a measure of the integrity of the actin cytoskeleton in living stamen hair cells, we demonstrated that AtFim1 protects actin filaments in these cells from Z. mays profilin (ZmPRO5)-induced depolymerization, in a dose-dependent manner. The apparent ability of AtFim1 to protect actin filaments in vivo from profilin-mediated depolymerization was confirmed by in vitro sedimentation assays. Our results indicate that AtFim1 is a calcium-independent, actin filament-crosslinking protein that interacts with the actin cytoskeleton in living plant cells.     

Enzyme-catalyzed detoxication reactions: mechanisms and stereochemistry

Enzyme catalyzed detoxication reactions are one of the primary defenses organisms have against chemical insult. This article reviews current chemical approaches to understanding the cooperative role of enzymes in the metabolism of foreign compounds. Emphasis is placed on chemical and stereochemical studies which help elucidate the mechanism of action and active-site topologies of the detoxication enzymes. The stereoselectivity of the cytochromes P-450 and flavin containing monooxygenases as well as the role of hemoglobin and lipid peroxidation in the primary metabolism of xenobiotics is discussed. Current knowledge of the mechanism and stereoselectivity of epoxide hydrolase is also presented. Three enzymes involved in secondary metabolism of xenobiotics, UDP-glucuronosyltransferase, sulfotransferase and glutathione S-transferase are discussed with particular emphasis on active site topology and cooperative participation with the enzymes of primary metabolism.     

Enzyme-catalyzed preparative peptide synthesis in frozen aqueous systems

α-Chymotrypsin (α-CT; EC 3.4.21.1) and papain (EC 3.4.22.2) were used as catalysts in preparative peptide synthesis in frozen aqueous systems. A special apparatus was constructed in order to enable shock freezing of large reaction volumes. Several hundred milligram of peptides including non-natural components were synthesized representing yields of 43% to 95%.     

Enzyme-catalyzed decomposition of dibenzoyl peroxide in organic solvents

Catalytic activity of catalase (CAT, EC 1.11.1.6), immobilized on carbon black NORIT and soot PM-100, with respect to decomposition of dibenzoyl peroxide (BPO) in non-aqueous media (acetonitrile and tetrachloromethane), was investigated with a quantitative UV-spectrophotometrical approach. Progress of the above reaction was controlled by selected kinetic parameters: the apparent Michaelis constant (Km(app)), the specific rate constant (k(sp)), the activation energy (Ea), the maximum reaction rate (Vmax), and the Arrhenius' pre-exponential factor (Z0). Conclusions on the tentative mechanism of the catalytic process observed were drawn from the calculated values of the Gibbs energy of activation (deltaG*), the enthalpy of activation (deltaH*), and entropy of activation (deltaS*).     

Enzyme-catalyzed synthesis of optically pure R(+)-phenylethanol

Summary A dehydrogenase catalyzing the NADPH-dependent reduction of acetophenone to R(+)-phenylethanol was found inLactobacillus kefir. A continuous conversion of 10 mM acetophenone with a yield of up to 90% was achieved in an enzyme-membrane-reactor with simultaneous NADPH-regeneration using glucose-6-phosphate and glucose-6-phosphate dehydrogenase. Enzyme-catalyzed oxidation of phenylethanol occurred with the R(+)-enantiomer only, whereas S(–) was not converted by the enzyme. The formation of R(+)-phenylethanol with an enantiomeric excess of 100% was confirmed by chiral HPLC.     

Enzyme-catalyzed synthesis of sugar-containing monomers and linear polymers

Commercially available proteases and lipases were screened for their ability to acylate regioselectively sucrose and trehalose with divinyladipic acid ester. Opticlean M375 (subtilisin from Bacillus licheniformis) was observed to form sucrose 1'-O-adipate and trehalose 6-O-adipate in anhydrous pyridine. Novozym-435 (lipase B from Candida antarctica) catalyzed the synthesis of sucrose 6, 6'-O-divinyladipate and trehalose 6, 6'-O-divinyladipate in acetone. These diesters were then employed as monomers in polycondensation reactions with various diols (aliphatic and aromatic) catalyzed by Novozym-435 in organic solvents to yield linear polyesters with M(w)'s up to 22,000 Da. Spectroscopic analysis confirmed that only the vinyl end groups of sugar esters reacted in the enzymatic polymerization with the diol, and not the internal sugar-adipate linkages. The two-step enzymatic strategy to yield sugar-based polyesters, which is the first report of its kind, results in higher molecular weights and faster reaction times than one-step enzymatic polyester synthesis.     

Determination of protein oligomerization state: two approaches based on glutaraldehyde crosslinking

Many biochemical and biophysical methods can be used to characterize the oligomerization state of proteins. One of the most widely used is glutaraldehyde crosslinking, mainly because of the minimum equipment and reagents required. However, the crosslinking procedures currently in use are impaired by the low specificity of the reagent, which can chemically bond any two amino groups that are close in space. Thus, extensive and time-consuming investigation of the reaction conditions is usually required. Here we describe two approaches based on glutaraldehyde that readily give reliable results.     

Mechanism based protein crosslinking of domains from the 6-deoxyerythronolide B synthase

The critical role of protein-protein interactions in the chemistry of polyketide synthases is well established. However, the transient and weak nature of these interactions, in particular those involving the acyl carrier protein (ACP), has hindered efforts to structurally characterize these interactions. We describe a chemo-enzymatic approach that crosslinks the active sites of ACP and their cognate ketosynthase (KS) domains, resulting in the formation of a stable covalent adduct. This process is driven by specific protein-protein interactions between KS and ACP domains. Suitable manipulation of the reaction conditions enabled complete crosslinking of a representative KS and ACP, allowing isolation of a stable, conformationally constrained adduct suitable for high-resolution structural analysis.     

Human protein aging: modification and crosslinking through dehydroalanine and dehydrobutyrine intermediates

Nonenzymatic post‐translational modification (PTM) of proteins is a fundamental molecular process of aging. The combination of various modifications and their accumulation with age not only affects function, but leads to crosslinking and protein aggregation. In this study, aged human lens proteins were examined using HPLC–tandem mass spectrometry and a blind PTM search strategy. Multiple thioether modifications of Ser and Thr residues by glutathione (GSH) and its metabolites were unambiguously identified. Thirty‐four of 36 sites identified on 15 proteins were found on known phosphorylation sites, supporting a mechanism involving dehydroalanine (DHA) and dehydrobutyrine (DHB) formation through β‐elimination of phosphoric acid from phosphoserine and phosphothreonine with subsequent nucleophilic attack by GSH. In vitro incubations of phosphopeptides demonstrated that this process can occur spontaneously under physiological conditions. Evidence that this mechanism can also lead to protein–protein crosslinks within cells is provided where five crosslinked peptides were detected in a human cataractous lens. Nondisulfide crosslinks were identified for the first time in lens tissue between βB2‐ & βB2‐, βA4‐ & βA3‐, γS‐ & βB1‐, and βA4‐ & βA4‐crystallins and provide detailed structural information on in vivo crystallin complexes. These data suggest that phosphoserine and phosphothreonine residues represent susceptible sites for spontaneous breakdown in long‐lived proteins and that DHA‐ and DHB‐mediated protein crosslinking may be the source of the long‐sought after nondisulfide protein aggregates believed to scatter light in cataractous lenses. Furthermore, this mechanism may be a common aging process that occurs in long‐lived proteins of other tissues leading to protein aggregation diseases.     

Enzyme-catalyzed gel proteolysis: an anomalous diffusion-controlled mechanism

Enzyme-catalyzed proteolysis of gelatin gels has been studied. We report a gel degradation rate varying as the square of the enzyme concentration. The diffusion motion of enzymes in the gel has been measured by two-photon fluorescence correlation spectroscopy and identified as being anomalously slow. These experimental results are discussed from a theoretical point of view and interpreted in terms of a diffusion-controlled mechanism for the gel degradation. These results make a step toward the understanding of enzyme-catalyzed gel degradation and give new insight on biological processes such as the action of metalloproteinases in the extracellular matrix involved in cellular invasion.