Laccase‐catalysed polymeric dye synthesis from plant‐derived phenols for potential application in hair dyeing: Enzymatic colourations driven by homo‐ or hetero‐polymer synthesis

Laccase efficiently catalyses polymerization of phenolic compounds. However, knowledge on applications of polymers synthesized in this manner remains scarce. Here, the potential of laccase-catalysed polymerization of natural phenols to form products useful in hair dyeing was investigated. All 15 tested phenols yielded coloured products after laccase treatment and colour diversity was attained by using mixtures of two phenolic monomers. After exploring colour differentiation pattern of 120 different reactions with statistical regression analysis, three monomer combinations, namely gallic acid and syringic acid, catechin and catechol, and ferulic acid and syringic acid, giving rise to brown, black, and red materials, respectively, were further characterized because such colours are commercially important for grey hair dyeing. Selected polymers could strongly absorb visible light and their hydrodynamic sizes ranged from 100 to 400 nm. Analyses of enzyme kinetic constants, liquid chromatography and electrospray ionization-mass spectrometry (ESI-MS) coupled with collision-induced dissociation MS/MS indicate that both monomers in reactions involving catechin and catechol, and ferulic acid and syringic acid, are coloured by heteropolymer synthesis, but the gallic acid/syringic acid combination is based on homopolymer mixture formation. Comparison of colour parameters from these three reactions with those of corresponding artificial homopolymer mixtures also supported the idea that laccase may catalyse either hetero- or homo-polymer synthesis. We finally used selected materials to dye grey hair. Each material coloured hair appropriately and the dyeing showed excellent resistance to conventional shampooing. Our study indicates that laccase-catalysed polymerization of natural phenols is applicable to the development of new cosmetic pigments.     

An evolutionary approach to enzyme kinetics: Optimization of ordered mechanisms

A theoretical investigation is presented which allows the calculation of rate constants and phenomenological parameters in states of maximal reaction rates for unbranched enzymic reactions. The analysis is based on the assumption that an increase in reaction rates was an important characteristic of the evolution of the kinetic properties of enzymes. The corresponding nonlinear optimization problem is solved taking into account the constraint that the rate constants of the elementary processes do not exceed certain upper limits. One-substrate-one-product reactions with two, three and four steps are treated in detail. Generalizations concern ordered uni-uni-reactions involving an arbitrary number of elementary steps. It could be shown that depending on the substrate and product concentrations different types of solutions can be found which are classified according to the number of rate constants assuming in the optimal state submaximal values. A general rule is derived concerning the number of possible solutions of the given optimization problem. For high values of the equilibrium constant one solution always applies to a very large range of the concentrations of the reactants. This solution is characterized by maximal values of the rate constants of all forward reactions and by non-maximal values of the rate constants of all backward reactions. Optimal kinetic parameters of ordered enzymic mechanisms with two substrates and one product (bi-uni-mechanisms) are calculated for the first time. Depending on the substrate and product concentrations a complete set of solutions is found. In all cases studied the model predicts a matching of the concentrations of the reactants and the corresponding Michaelis constants, which is in good accordance with the experimental data. It is discussed how the model can be applied to the calculation of the optimal kinetic design of real enzymes.     

Structure of chalcone synthase and the molecular basis of plant polyketide biosynthesis.

Chalcone synthase (CHS) is pivotal for the biosynthesis of flavonoid antimicrobial phytoalexins and anthocyanin pigments in plants. It produces chalcone by condensing one p-coumaroyl- and three malonyl-coenzyme A thioesters into a polyketide reaction intermediate that cyclizes. The crystal structures of CHS alone and complexed with substrate and product analogs reveal the active site architecture that defines the sequence and chemistry of multiple decarboxylation and condensation reactions and provides a molecular understanding of the cyclization reaction leading to chalcone synthesis. The structure of CHS complexed with resveratrol also suggests how stilbene synthase, a related enzyme, uses the same substrates and an alternate cyclization pathway to form resveratrol. By using the three-dimensional structure and the large database of CHS-like sequences, we can identify proteins likely to possess novel substrate and product specificity. The structure elucidates the chemical basis of plant polyketide biosynthesis and provides a framework for engineering CHS-like enzymes to produce new products.     

Combining sequence-specific probes and DNA binding dyes in real-time PCR for specific nucleic acid quantification and melting curve analysis

Currently, in real-time PCR, one often has to choose between using a sequence-specific probe and a nonspecific double-stranded DNA (dsDNA) binding dye for the detection of amplified DNA products. The sequence-specific probe has the advantage that it only detects the targeted product, while the nonspecific dye has the advantage that melting curve analysis can be performed after completed amplification, which reveals what kind of products have been formed. Here we present a new strategy based on combining a sequence-specific probe and a nonspecific dye, BOXTO, in the same reaction, to take the advantage of both chemistries. We show that BOXTO can be used together with both TaqMan probes and locked nucleic acid (LNA) probes without interfering with the PCR. The probe signal reflect formation of target product, while melting curve analysis of the BOXTO signal reveals primer-dimer formation and the presence of any other anomalous products.     

Enzyme induction and repression in anabolic and catabolic pathways

Summary Microorganisms have evolved enzymes which catalyze a large number of reactions in the sequences to form essential cellular constituents and liberate energy and carbon for cellular processes. Regulation of the use of energy and of the monomeric cellular precursors to the synthesis of those enzymes required under changing environmental conditions depends on the one hand on the level of end products of a reaction sequence and on the other upon the presence of the first, or early members of a reaction sequence. These cases in turn represent product repression and substrate, or substrate like, induction of enzyme formation. Though the repression system has generally been considered to operate in anabolic and the induction system in catabolic processes, the experiments presented demonstrate a role for both types of control in formation of biosynthetic and peripheral pathway enzymes. The induction of biosynthetic enzymes is shown in Pseudomonas putida, and organism with three clusters of genes for the tryptophan pathway. The repression of degradative enzymes is shown in an extended pathway of peripheral oxidation of terpenoid compounds. The enzymes for steps following conversion of neutral to non-essential acidic products are repressed as well as enzymes beyond convergence with isobutyrate formation and conversion to the succinyl and propionyl intermediates.Dedicated to C. B. van Niel on the occasion of his 70th birthday. Supported in part by grant G24037 from the National Science Foundation.     

A Kinetic Analysis of Coupled (or Auxiliary) Enzyme Reactions

As a case study, we consider a coupled (or auxiliary) enzyme assay of two reactions obeying the Michaelis–Menten mechanism. The coupled reaction consists of a single-substrate, single-enzyme non-observable reaction followed by another single-substrate, single-enzyme observable reaction (indicator reaction). In this assay, the product of the non-observable reaction is the substrate of the indicator reaction. A mathematical analysis of the reaction kinetics is performed, and it is found that after an initial fast transient, the coupled reaction is described by a pair of interacting Michaelis–Menten equations. Moreover, we show that when the indicator reaction is fast, the quasi-steady-state dynamics are governed by three fast variables and one slow variable. Timescales that approximate the respective lengths of the indicator and non-observable reactions, as well as conditions for the validity of the Michaelis–Menten equations, are derived. The theory can be extended to deal with more complex sequences of enzyme-catalyzed reactions.     

Role of Homocysteine Synthetase in an Alternate Route for Methionine Biosynthesis in Saccharomyces cerevisiae

In vivo studies have shown that, in the absence of homoserine-O-transacetylase activity (locus met(2)), the C(4)-carbon moiety of ethionine is utilized (provided the ethionine resistance gene eth-2r is present) by methionine auxotrophs, except for met(8) mutants (homocysteine synthetase-deficient). Concomitant utilization of sulfur and methyl group from methylmercaptan or S-methylcysteine has been demonstrated. In the absence of added methylated intermediates, the methyl group of methionine formed from ethionine is derived from serine. In vitro studies with crude extracts of Saccharomyces cerevisiae have demonstrated that this synthesis of methionine occurs by the following reactions: CH(3)-SH + ethionine right harpoon over left harpoon methionine + C(2)H(5)SH and S-methylcysteine + ethionine right harpoon over left harpoon methionine + S-ethylcysteine. In the forward direction, the second product of the second reaction was shown to be S-ethylcysteine; this reaction has also been found reversible, leading to ethionine formation. Genetic and kinetic data have shown that homocysteine synthetase catalyzes these two reactions, at 0.3% of the rate it catalyzes direct homocysteine synthesis: O-Ac-homoserine + Na(2)S --> homocysteine + acetate. The three reactions are lost together in a met(8) mutant and are recovered to the same extent in spontaneous prototrophic revertants from this strain. Methionine-mediated regulation of enzyme synthesis affects the three activities and is modified to the same extent by the presence of the recessive allele (eth-2r) of the regulatory gene eth-2. Affinities of the enzyme for substrates of both types of reactions are of the same order of magnitude. Moreover, ethionine, the substrate of the second reaction, inhibits the third reaction, whereas O-acetyl-homoserine, the substrate of the third reaction, inhibits the second reaction. An enzymatic cleavage of S-methylcysteine, leading to methylmercaptan production, has been shown to occur in crude yeast extracts. It is concluded that the enzyme homocysteine synthetase participates in the two alternate pathways leading to methionine biosynthesis in S. cerevisiae, one involving O-acetyl-homoserine and H(2)S, the other involving the 4-carbon chain of ethionine and a mercaptyl donor. Participation of the two types of reactions catalyzed by homocysteine synthetase, in in vivo methionine synthesis, has been shown to occur in a met(2) partial revertant.     

FLP site-specific recombinase of yeast 2-micron plasmid. Topological features of the reaction

The 2-micron plasmid of the yeast Saccharomyces cerevisiae encodes a site-specific recombinase (FLP) that promotes inversion across a unique site contained in each of the 599-base-pair inverted repeats of the plasmid. We have studied the topological changes generated in supercoiled substrates after exposure to the purified FLP protein in vitro. When a supercoiled substrate bearing two FLP target sequences in inverse orientation is treated with FLP, the products are multiply knotted structures that arise as a result of random entrapment of interdomainal supercoils. Likewise, a supercoiled substrate bearing two target sequences in direct orientation yields multiply interlocked catenanes as the product. Both types of substrate seem to be able to undergo repeated rounds of recombination that result in products of further complexity. The FLP protein also acts as a site-specific topoisomerase during the recombination reaction.     

Enzymatic ligation assisted by nucleases: simultaneous ligation and digestion promote the ordered assembly of DNA

This protocol describes a method for the one-tube preparative-scale assembly of a specific DNA molecule, the enzymatic ligation assisted by nucleases (ELAN) technique. DNA fragments in ligation reactions are capable of combining to produce numerous products. The ELAN method uses judicious choice of restriction enzyme sites coupled with simultaneous digestion and ligation reactions to create just one product, by converting off-pathway products back into substrate. The experimental parameters critical for a successful ELAN reaction are discussed, and the ordered, one-tube assembly of four DNA fragments in the presence of eight restriction enzymes is demonstrated. This technique will be useful to those performing gene construction, DNA computing, biophysics and even standard molecular cloning. Starting with reactant fragments, the protocol takes 4-16 h to produce nanogram to microgram yields, depending on the complexity of the reaction.     

A novel reactive perstraction system based on liquid-core microcapsules applied to lipase-catalyzed biotransformations

A novel reactive perstraction system has been developed based on liquid-core capsules, involving an enzyme-catalyzed reaction coupled with simultaneous in situ product recovery. Lipase-catalyzed reactions, hydrolysis of triprionin and nitrophenyl laurate, were selected to test the system and demonstrate the feasibility of immobilization of enzymes to the membranes of liquid-core capsules and the ability to extract hydrophobic products of the reaction within the capsule core. The lipase from Candida rugosa was immobilized to the microcapsules by adsorption and by covalent binding through activation with glutaraldehyde. In both cases improved temperature and operational stability were achieved. Both types of immobilization resulted in a basic shift of the pH optimum for activity, from 7.5 to 9.0. The presence of an organic phase within the capsule core allowed direct product separation and lead to a decrease in product inhibition of the lipase-catalyzed reaction.     

Ultrastructural electron probe X-ray microanalytical reaction product identification of three different enzymes in the same mouse resident peritoneal macrophage

Simultaneous cytochemical enzyme localization procedures for peroxidase (PO) plus acid phosphatase (AcP-ase) and/or aryl sulphatase (AS) have been investigated at the ultrastructural (EM) level. Electron probe X-ray microanalysis (EPMA) will identify and differentiate the reaction products. Dual reaction product localization of PO plus AcP-ase or alternatively PO plus AS have been obtained in the same mouse resident peritoneal macrophage. This has been acquired by first performing a PO-reaction followed by AcP-ase or followed by AS. In both cases PO-related reaction products (PODAB/Os or PODAB/Pt) were localized in nuclear envelope (NE) and rough endoplasmic reticulum (RER). Cells were identified by this reaction product as resident macrophages. Reaction products from the AcP-ase related cerium (AcP-aseCe), localized in lysosomes have been identified and differentiated from the PO-related osmium containing products. Similarly AS related barium (ASBa), localized in lysosomal structures and (R)ER was identified and differentiated. Triple reaction product localization of PO followed by AcP-ase plus AS could also be obtained. In this case, PO-related platinum containing reaction products (PODAB/Pt or PODAB/Os) in NE and RER has been identified and differentiated from the AcP-ase related lysosomal cerium (AcP-aseCe) and the AS related barium localized in lysosomal and (R)ER structures. Reversing the sequences in both dual cytochemical procedures: AcP-aseCe or ASBa followed by PODAB/Os (or PODAB/Pt) resulted in AcP-aseCe or ASBa activity related reaction products only. Reversing the sequence in the triple reaction procedures (ASBa followed by AcP-aseCe) resulted in the absence of the barium containing reaction products. By application of OsO4 postfixation with aminotriazole (ATR) additives the detrimental effects upon the various precipitates have been confirmed. In LM studies, using rat intestine and non-metal identification reactions for two of the enzymes (pararosaniline for AcP-ase, DAB for peroxidase), the influences of the metal ions used in EM were tested on the appearance of the coloured reaction products. Cerium ions used in EM for detection of AcP-aseCe activity have been shown to influence the PODAB visibility in LM and EM experiments. From the AS reaction media components neither barium ions nor p-nitro catachol sulphate influenced the LM visibility of the PO reaction.     

Linking-number changes in the DNA substrate during Cre-mediated loxP site-specific recombination

We have examined the linking-number changes that occur during phage P1 Cre-mediated recombination in vitro between two loxP sites. Such recombination reactions can be divided into three types: intramolecular inversion, in which recombination occurs between two loxP sites in opposite orientations on the same DNA substrate; intramolecular excision, where recombination occurs between two loxP sites that are in the same orientation on the DNA substrate; and intermolecular recombination, which occurs between two loxP sites on separate DNA molecules. Our results indicate that inversion changes the linking number of the substrate DNA by two topological turns. With a negatively supercoiled substrate, the product is changed by +2 turns. A relaxed substrate yields products that have been changed by either +2 or -2 turns. For intermolecular reactions, the sum of the linking numbers of each of the two starting circles is equal to the linking number of the dimer circle generated by recombination, and no change occurs in linking number. For intramolecular excision reactions, the data are most consistent, with no change in linking number during recombination. These results are discussed in terms of models for alignment and synapsis of the recombining sites and the mechanism of strand exchange.     

Hydrolysis of human high-molecular-mass kininogen by human plasma kallikrein. Proposal of a new model concept for the course of reaction in presence and absence of C1(-)-inhibitor

Hydrolysis of high-molecular-mass kininogen was studied by following the changes in the amounts of substrate, intermediates and products as a function of time using quantitative polyacrylamide-gel electrophoresis (silver staining). The experimental data was analysed on the basis of the concept that the overall reaction is composed of three hydrolysis reactions, two positional-change processes of intermediates at the active site, and two product-substrate exchange processes. It is proposed C1(-)-inhibitor to form two types of complexes with kallikrein, one with non-covalent and one with covalent bonds. With an adequately chosen set of reaction-partner concentrations and four different kinds of experimental conditions with respect to kininogen and inhibitor addition to kallikrein, the following results were obtained: 1) Non-covalently bound inhibitor has no effect on the first and the second hydrolysis reaction, but efficiently interferes with the third hydrolysis reaction; 2) Nicked kininogen (first intermediate; one of the two bradykinin bonds split) for the second bond to be hydrolysed undergoes a positional change during which it remains strongly bound to the enzyme, never exchanges with kininogen, and is not displaced by non-covalently bound inhibitor; 3) Intermediate kinin-free kininogen (second intermediate; both bradykinin bonds split and bradykinin released) prior to turning over into stable kinin-free kininogen (final product; histidine-rich fragment split off and released) undergoes a positional change involving dissociation and reassociation so that non-covalently bound inhibitor finds access to the active site; 4) Intermediate kinin-free kininogen to sustain multiple turnovers exchanges with kininogen via a stable complex of such structure that during this process non-covalently bound inhibitor cannot or can only slightly interfere; 5) Stable kinin-free kininogen to sustain multiple turnovers exchanges with intermediate kinin-free kininogen via free enzyme with the effect that non-covalently bound inhibitor efficiently interferes; 6) As hydrolysis proceeds more and more inhibitor becomes covalently bound, gradually leading to complete inactivation of the enzyme.     

Functional analysis of eubacterial diterpene cyclases responsible for biosynthesis of a diterpene antibiotic,terpentecin

Eubacterial diterpene cyclase genes had previously been cloned from a diterpenoid antibiotic terpentecin producer (Dairi, T., Hamano, Y., Kuzuyama, T., Itoh, N., Furihata, K., and Seto, H. (2001) J. Bacteriol. 183, 6085-6094). Their products, open reading frame 11 (ORF11) and ORF12, were essential for the conversion of geranylgeranyl diphosphate (GGDP) into terpentetriene (TTE) that had the same basic skeleton as terpentecin. In this study, functional analyses of these two enzymes were performed by using purified recombinant enzymes. The ORF11 product converted GGDP into a cyclized intermediate, terpentedienol diphosphate (TDP), which was then transformed into TTE by the ORF12 product. Interestingly, the ORF12 product directly catalyzed the conversion of GGDP into three olefinic compounds. Moreover, the ORF12 product utilized farnesyl diphosphate as a substrate to give three olefinic compounds, which had the same structures as those formed from GGDP with the exception of the chain lengths. These results suggested that the ORF11 product with a DXDD motif converted GGDP into TDP by a protonation-initiated cyclization and that the ORF12 product with a DDXXD motif completed the transformation of TDP to the olefin, terpentetriene by an ionization-initiated reaction followed by deprotonation. The kinetics of the ORF12 product indicated that the affinity for TDP and GGDP were higher than that of farnesyl diphosphate and that the relative activity of the reaction converting TDP into TTE was highest among the reactions using TDP, GGDP, or farnesyl diphosphate as the substrate. These results suggested that an actual reaction catalyzed by the ORF12 was the conversion of TDP into TTE in vivo.     

Description of the ratios between metabolite concentrations in a polyenzyme system

It was demonstrated that the relations between substrate and product concentrations for a reaction catalyzed by michaelian enzyme incorporated in a multienzyme system can be graphically represented by a diverging set of straight lines intersecting in one point, the flux velocity being treated as a parameter. A competitive inhibitor shifts the intersection point along the line of equilibrium state. The relations between the concentrations of more than two reagents are represented by a set of equivelocity surfaces. The relations between substrate and product concentrations for a kinetically cooperative reaction conforming to the graphical representation by the second--order curves were analyzed. The stability criterion was obtained for a multienzyme system with the first enzyme allosterically regulated by products of subsequent reactions.     

Enzymatic assays for NAD-dependent deacetylase activities

The NAD-dependent deacetylases are a new class of enzymes responsible for the removal of acetyl groups from lysines on proteins. Instead of water, the NAD-dependent deacetylases use a highly reactive ADP-ribose intermediate as a recipient for the acetyl group. The products of the reaction are nicotinamide, acetyl-ADP-ribose, and a deacetylated substrate. Many assays have been developed for the measurement of NAD-dependent deacetylase activity. In this review we present assays based on each of the two reactions catalyzed by these enzymes, deacetylation and NAD hydrolysis. First we describe methods for the production of acetylated protein and peptide substrates for use in deacetylation reactions. Then we describe four methods for assaying deacetylation, three of which directly measure the loss of acetyl groups from a protein or peptide substrate, and one that measures acetate production. We also describe two indirect methods for following enzyme activity, NAD hydrolysis and a novel NAD-nicotinamide exchange reaction. Finally, a quantitative method using a monoacetylated peptide as a substrate and HPLC to measure products is described.     

The inhibition of pepsin-catalysed reactions by structural and stereochemical product analogues

1. The inhibition of pepsin-catalysed hydrolysis of N-acetyl-l-phenylalanyl-l-phenylalanylglycine by products and product analogues was studied. 2. Inhibitors of the l-configuration give rise to linear non-competitive inhibition, whereas those of the d-configuration show linear competitive behaviour. 3. Non-competitive inhibition by the product N-acetyl-l-phenylalanine indicates an ordered release of products, which supports a common mechanism (involving an ;amino-enzyme') for pepsin-catalysed transpeptidation and hydrolysis reactions. 4. The differences in the types of inhibition caused by product analogues of the l- and d-series emphasize the stereospecificity of the binding of these inhibitors to free enzyme and to the putative amino-enzyme intermediate. 5. The results suggest that it is the anion of the acyl product that is released first in the hydrolytic reaction (see Kitson & Knowles, 1971).     

Mechanism of action of adenosylcobalamin: 3-fluoro-1,2-propranediol as substrate for propanediol dehydrase--mechanistic implications.

3-Fluro-1,2-propanediol has been found to be a substrate for propanediol dehydrase and has very similar binding and catalytic constants compared to the natural substrate. The only isolable products of the reaction are acrolein and inorganic fluoride; with 3-fluoro-3,3-dideuterio-1,2-propanediol as substrate, only 3,3-dideuterioacrolein is obtained. These results indicate that the primary product of the reaction is 3-fluoropropionaldehyde which spontaneously loses hydrogen fluoride to yield acrolein. The similar kinetic parameters for the fluorinated as compared to the normal substrate suggest that significant charge does not develop on the fluorinated or, by implication, the natural substrate during any rate-limiting steps of the reaction. These results support a radical, as contrasted to an ionic pathway for reactions involving adenosylcobalamin and diol dehydrase.     

Substrate inactivation of enzymes in vitro and in vivo

Some enzymes are inactivated by their natural substrates during catalytic turnover, limiting the ultimate extent of reaction. These enzymes can be separated into three broad classes, depending on the mechanism of the inactivation process. The first type is enzymes which use molecular oxygen as a substrate. The second type is inactivated by hydrogen peroxide, which is present either as a substrate or a product, and are stabilized by high catalase activity. The oxidation of both types of enzymes shares common features with oxidation of other enzymes and proteins. The third type of enzyme is inactivated by non-oxidative processes, mainly reversible loss of cofactors or attached groups. Sub classes are defined within each broad classification based on kinetics and stoichiometry. Reaction-inactivation is in part a regulatory mechanism in vivo, because specific proteolytic systems give rapid turnover of such labelled enzymes. The methods for enhancing the stability of these enzymes under reaction conditions depends on the enzyme type. The kinetics of these inactivation reactions can be used to optimize bioreactor design and operation.