Pericyclic reactions catalyzed by chorismate-utilizing enzymes

One of the fundamental questions of enzymology is how catalytic power is derived. This review focuses on recent developments in the structure--function relationships of chorismate-utilizing enzymes involved in siderophore biosynthesis to provide insight into the biocatalysis of pericyclic reactions. Specifically, salicylate synthesis by the two-enzyme pathway in Pseudomonas aeruginosa is examined. The isochorismate-pyruvate lyase is discussed in the context of its homologues, the chorismate mutases, and the isochorismate synthase is compared to its homologues in the MST family (menaquinone, siderophore, or tryptophan biosynthesis) of enzymes. The tentative conclusion is that the activities observed cannot be reconciled by inspection of the active site participants alone. Instead, individual activities must arise from unique dynamic properties of each enzyme that are tuned to promote specific chemistries.     

Kinetic modeling of interesterification reactions catalyzed by immobilized lipase

Kinetic data for lipase-catalyzed interesterification reactions between free fatty acids and triglycerides were collected and the dynamics of the interesterification reactions were successfully modeled using tow rate experssions requiring a total of five adjustable parameters. One rate expression describes the disappearance of the free fatty acid (octanoic or linolenic acid), and the second describes the rate of release of fatty acid residues from the triglycerides (olive oil or milkfat). This model is able to account for the effects of the concentration of all chemical species participating in interesterification throughout the entire reaction. When the data for both milkfat and olive oil were subjected to nonlinear regression analyses using the same mathematical model, the parameter estimates for both systems were comparable. In addition to reproducing the tendencies observed experimentally, simulations of the interesterification system under a variety of initial conditions provided insight into the effects of several reaction variables which could not be examined experimentally. Among the most significant findings of the simulation work are (1) there is a limit beyond which increasing the initial concentration of water produces no further increase in the initial rate of the interesterification reaction; (2) an increase in the initial concentration of lower glycerides produces a concomitant increase in the rate of the interesterification reaction; (3) the free fatty acids inhibit the rate of hydrolysis of the fatty acid residues of the triglycerides; (4) there is a limit beyond which increasing the initial concentration of triglycerides produces no significant increase in the rate of either the hydrolysis reaction or the interesterification reaction. (c) 1994 John Wiley & Sons, Inc.     

Gas phase transesterification reactions catalyzed by lipolytic enzymes

Porcine pancreatic lipase and Fusarium solani cutinase were used to catalyze transesterification reactions between methyl propionate, ethyl propionate, and a series of primary alcohols at high temperatures in a continuous packed-bed gas-solid reactor, in which the solid phase is composed of the enzyme and the substrates and products are in a gaseous form. In this type of system, enzyme activity was found to depend essentially on the water activity (A(w)) of the enzyme preparation.     

Complex reactions catalyzed by cytochrome P450 enzymes

Cytochrome P450 (P450) enzymes are some of the most versatile redox proteins known. The basic P450 reactions include C-hydroxylation, heteroatom oxygenation, heteroatom release (dealkylation), and epoxide formation. Mechanistic explanations for these reactions have been advanced. A number of more complex P450 reactions also occur, and these can be understood largely in the context of the basic chemical mechanisms and subsequent rearrangements. The list discussed here updates a 2001 review and includes chlorine oxygenation, aromatic dehalogenation, formation of diindole products, dimer formation via Diels-Alder reactions of products, ring coupling and also ring formation, reductive activation (e.g., aristolochic acid), ring contraction (piperidine nitroxide radical), oxidation of troglitazone, cleavage of amino oxazoles and a 1,2,4-oxadiazole ring, bioactivation of a dihydrobenzoxathiin, and oxidative aryl migration.     

Asymmetric Stetter reactions catalyzed by thiamine diphosphate-dependent enzymes

The intermolecular asymmetric Stetter reaction is an almost unexplored transformation for biocatalysts. Previously reported thiamine diphosphate (ThDP)-dependent PigD from Serratia marcescens is the first enzyme identified to catalyze the Stetter reaction of α,β-unsaturated ketones (Michael acceptor substrates) and α-keto acids. PigD is involved in the biosynthesis of the potent cytotoxic agent prodigiosin. Here, we describe the investigation of two new ThDP-dependent enzymes, SeAAS from Saccharopolyspora erythraea and HapD from Hahella chejuensis. Both show a high degree of homology to the amino acid sequence of PigD (39 and 51 %, respectively). The new enzymes were heterologously overproduced in Escherichia coli, and the yield of soluble protein was enhanced by co-expression of the chaperone genes groEL/ES. SeAAS and HapD catalyze intermolecular Stetter reactions in vitro with high enantioselectivity. The enzymes possess a characteristic substrate range with respect to Michael acceptor substrates. This provides support for a new type of ThDP-dependent enzymatic activity, which is abundant in various species and not restricted to prodigiosin biosynthesis in different strains. Moreover, PigD, SeAAS, and HapD are also able to catalyze asymmetric carbon–carbon bond formation reactions of aldehydes and α-keto acids, resulting in 2-hydroxy ketones.     

Kinetics and mechanisms of reactions catalyzed by immobilized lipases*

This review focuses on the kinetics of several modes of immobilization of lipases, on the mechanisms of reactions of activation of immobilized lipases, and on the kinetics and mechanisms of reactions catalyzed by immobilized lipases. A comprehensive overview of the state of the art pertaining to structural features of lipases is provided as an aid to understand immobilization, interfacial activation, and catalytic performance. General rate expressions are duly derived; more frequent simplifying assumptions are stated and the results thereof listed. Physicochemical and statistical significance of parameters in rate expressions fitted to experimental data are also discussed whenever possible.     

Adenosylation reactions catalyzed by the radical S-adenosylmethionine superfamily enzymes

The radical S-adenosylmethionine (SAM) superfamily enzymes reductively cleave SAM to produce a highly reactive 5ˊ-deoxyadenosyl (dAdo) radical, which in most cases abstracts a hydrogen from the substrate and initiates highly diverse reactions. In rare cases, the dAdo radical can add to a sp² carbon to result in the production an adenosylated product. These radical SAM-dependent adenosylation reactions are present in natural product biosynthetic pathways and can be achieved by using unnatural substrate analogs containing olefin or aryl moieties. This Opinion provides a focused perspective on this emerging type of biochemistry and discusses its potential use in bioengineering and biocatalysis.     

Theory of the kinetics of reactions catalyzed by enzymes attached to membranes

A theoretical treatment has been worked out for the kinetics of solid-supported enzyme systems, with diffusive and electrostatic effects taken into account. A utilization factor, defined as the ratio of the actual reaction rate to the rate of substrate consumption in the outer solution, is defined, and equations to evaluate the utilization factor are given for five kinetic conditions: (a) Michaelis-Menten behavior, (b) substrate inhibition, (c) product inhibition (competitive), (d) product inhibition (noncompetitive), and (e) product inhibition (anticompetitive). When the solid-supported enzymes obey a Michaelis-Menten relationship, an equation for the apparent Michaelis constant is given and a criterion for insignificant diffusion effects is shown. A substrate-inhibited enzyme reaction may display multiple steady-state behavior, and a criterion for uniqueness is presented. In the case of product-inhibited enzyme reactions, the utilization factor is always less than that which corresponds to a Michaelis-Menten relationship. Equations to evaluate the apparent Michaelis and inhibition constants are given.     

Progress curves of reactions catalyzed by unstable enzymes. A theoretical approach

When an enzyme is incubated with its substrate, the rate of catalysis will decline with time due to the combined effects of substrate utilization and product accumulation. These effects will be superimposed upon a progressive loss of catalytic activity if the enzyme is unstable, either spontaneously or as a result of an added reagent. In this report, the effect of enzyme inactivation on the progress curve for an enzyme-catalyzed reaction is considered. It is shown that under most circumstances catalysis will stop before the substrate is totally exhausted and that the amount of substrate remaining is related to the inactivation rate constants for various intermediates on the catalytic pathway. A graphical method for estimating these inactivation rate constants is suggested for several situations, including one which encompasses the effect of a suicide substrate. Expressions for the half time of the reaction are also given for some special cases.     

Esterification reactions catalyzed by lipases immobilized in organogels: effect of temperature and substrate diffusion

Rhizomucor miehei lipase was immobilized in hydroxy(propylmethyl) cellulose or agar gels containing lecithin or AOT microemulsions. The effect of the diffusion of substrates and products to this catalyst was studied, as well as the effect of temperature on the initial rate of ester synthesis. The composition of the gel affects the reaction rate due to mass transport phenomena. The apparent activation energies were higher for the systems based on agar, independently of the microemulsion used, and lower for the systems based on AOT microemulsions, independently of the polymer used.     

Pyrogallol-to-phloroglucinol conversion and other hydroxyl-transfer reactions catalyzed by cell extracts of Pelobacter acidigallici.

Permeabilized cells and cell extracts of Pelobacter acidigallici catalyzed the conversion of pyrogallol (1,2,3-trihydroxybenzene) to phloroglucinol (1,3,5-trihydroxybenzene) in the presence of 1,2,3,5-tetrahydroxybenzene. Pyrogallol consumption by resting cells stopped after lysis by French press or mild detergent (cetyltrimethylammonium bromide [CTAB]) treatment. Addition of 1,2,3,5-tetrahydroxybenzene to the assay mixture restored pyrogallol consumption and led to stoichiometric phloroglucinol accumulation. The stoichiometry of pyrogallol conversion to phloroglucinol was independent of the amount of tetrahydroxybenzene added. The tetrahydroxybenzene concentration limited the velocity of the transhydroxylation reaction, which reached a maximum at 1.5 mM tetrahydroxybenzene (1 U/mg of protein). Transhydroxylation was shown to be reversible. The equilibrium constant of the reaction was determined, and the free-energy change (delta G degree') of phloroglucinol formation from pyrogallol was calculated to be -15.5 kJ/mol. Permeabilized cells and cell extracts also catalyzed the transfer of hydroxyl moieties between other hydroxylated benzenes. Tetrahydroxybenzene and hydroxyhydroquinone participated as hydroxyl donors and as hydroxyl acceptors in the reaction, whereas pyrogallol, resorcinol, and phloroglucinol were hydroxylated by both donors. A novel mechanism deduced from these data involves intermolecular transfer of the hydroxyl moiety from the cosubstrate (1,2,3,5-tetrahydroxybenzene) to the substrate (pyrogallol), thus forming the product (phloroglucinol) and regenerating the cosubstrate.     

Immobilization of cell-associated enzymes by entrapment in polymethacrylamide beads

Immobilization of E. coil, Aspergillus niger and Aspergillus japonicus was accomplished by entrapment in polymethacrylamide beads via a two-phase polymerization using soybean oil as the suspension medium. The immobilized E. coli cells containing penicillin G acylase possessed a higher activity than that reported in the literature. The mmobilized mycelia of Aspergillus sp. containing β-fructofuranosidase were effective for catalyzing the formation of fructooligosaccharides.     

Kinetically controlled equilibria. The perturbation of hydrolysis equilibria in reactions catalyzed by alpha-chymotrypsin immobilized on charges supports

The hydrolysis and synthesis of N-Acetyl-I-tyrosine-ethyl-ester catalyzed by α-chymotrypsin immobilized in polymeric supports (Sephadex), with positive or negative stationary charges has been studied. Charged matrices perturbed the equilibrium (at pH 9.0), so that no complete hydrolysis was observed in the bulk solution and ester could be synthetized from acid and alcohol. The change is due to dipole orientation energies in the electric double layer where the reactions are catalyzed. This represents a situation where the equilibrium in the system is kinetically controlled by the equilibrium in a sub-system (here the electric double layer).     

Continuous penicillin production by Penicillium chrysogenum immobilized in calcium alginate beads

Summary A high penicillin-producing Penicillium chrysogenum strain immobilized in calcium alginate beads was used for continuous penicillin fermentation in a bubble column and in a conical bubble fermentor. The fermentation was limited by the growth rate, dilution rates and the stability of the alginate beads. The immobilized cells lost their ability to produce penicillin in the bubble column after 48 h from beginning of the continuous fermentation. In the conical bubble fermentor the immobilized cells remained active for more than 7 days. This bioreactor ensured a good distribution of nutrients and oxygen as well as a higher mechanical stability of the alginate beads.