On the relationship between affinity for molecular hydrogen and the physiological directionality of hydrogenases.

The physiological significance of the generic reaction H(2)<-->2[H] is not always clear because hydrogenases may function in the breakdown of molecular hydrogen or in its synthesis or in both directions. Fe-hydrogenases have nevertheless been most often associated with proton reduction and NiFe-hydrogenases with hydrogen oxidation. A re-determination of the K(M) of H(2) oxidation by Pyrococcus furiosus NiFe-hydrogenase-I and by Desulfovibrio vulgaris Fe-hydrogenase suggests that affinity for hydrogen has been seriously underestimated and that the kinetics of hydrogen activation in relation to the directionality of hydrogenases should be re-evaluated.

The covalent structure of the small subunit from Pseudomonas putida amine dehydrogenase reveals the presence of three novel types of internal cross-linkages, all involving cysteine in a thioether bond.

Pseudomonas putida contains an amine dehydrogenase that is called a quinohemoprotein as it contains a quinone and two hemes c as redox active groups. Amino acid sequence analysis of the smallest (8.5 kDa), quinone-cofactor-bearing subunit of this heterotrimeric enzyme encountered difficulties in the interpretation of the results at several sites of the polypeptide chain. As this suggested posttranslational modifications of the subunit, the structural genes for this enzyme were determined and mass spectrometric de novo sequencing was applied to several peptides obtained by chemical or enzymatic cleavage. In agreement with the interpretation of the X-ray electronic densities in the diffraction data for the holoenzyme, our results show that the polypeptide of the small subunit contains four intrachain cross-linkages in which the sulfur atom of a cysteine residue is involved. Two of these cross-linkages occur with the beta-carbon atom of an aspartic acid, one with the gamma-carbon atom of a glutamic acid and the fourth with a tryptophanquinone residue, this adduct constituting the enzyme's quinone cofactor, CTQ. The thioether type bond in all four of these adducts has never been found in other proteins. CTQ is a novel cofactor in the series of the recently discovered quinone cofactors.

Influence of the ethanol and glucose supply rate on the rate and enantioselectivity of 3-oxo ester reduction by baker's yeast.

Baker's-yeast-mediated reductions of ketones hold great potential for the industrial production of enantiopure alcohols. In this article we describe the stoichiometry and kinetics of asymmetric ketone reduction by cell suspensions of bakers' yeast (Saccharomyces cerevisiae). A system for quantitative analysis of 3-oxo ester reduction was developed and allowed construction of full mass and redox balances as well as determination of the influence of different process parameters on aerobic ketone reduction. The nature of the electron donor (ethanol or glucose) and its specific consumption rate by the biomass (0-1 mol.kg dw(-1).h(-1)) affected the overall stoichiometry and rate of the process and the final enantiomeric excess of the product. Excess glucose as the electron donor, i.e. a very high consumption rate of glucose, resulted in a high rate of alcoholic fermentation, oxygen consumption, and biomass formation and therefore causing low efficiency of glucose utilization. Controlled supply of the electron donor at the highest rates applied prevented alcoholic fermentation but still resulted in biomass formation and a high oxygen requirement, while low rates resulted in a more efficient use of the electron donor. Low supply rates of ethanol resulted in biomass decrease while low supply rates of glucose provided the most efficient strategy for electron donor provision and yielded a high enantiomeric excess of ethyl (S)-3-hydroxybutanoate. In contrast to batchwise conversions with excess glucose as the electron donor, this strategy prevented by-product formation and biomass increase, and resulted in a low oxygen requirement.

Enzymes involved in the glycidaldehyde (2,3-epoxy-propanal) oxidation step in the kinetic resolution of racemic glycidol (2,3-epoxy-1-propanol) by Acetobacter pasteurianus.

It is already known that kinetic resolution of racemic glycidol (2,3-epoxy-1-propanol) takes place when Acetobacter pasteurianus oxidizes the compound to glycidic acid (2,3-epoxy-propionic acid) with glycidaldehyde (2,3-epoxy-propanal) proposed to be the transient seen in this conversion. Since inhibition affects the feasibility of a process based on this conversion in a negative sense, and the chemical reactivity of glycidaldehyde predicts that it could be the cause for the phenomena observed, it is important to know which enzyme(s) oxidise(s) this compound. To study this, rac.- as well as (R)-glycidaldehyde were prepared by chemical synthesis and analytical methods developed for their determination. It appears that purified quinohemoprotein alcohol dehydrogenase (QH-ADH type II), the enzyme responsible for the kinetic resolution of rac.-glycidol, also catalyses the oxidation of glycidaldehyde. In addition, a preparation exhibiting dye-linked aldehyde dehydrogenase activity for acetaldehyde, most probably originating from molybdohemoprotein aldehyde dehydrogenase (ALDH), which has been described for other Acetic acid bacteria, oxidised glycidaldehyde as well with a preference for the (R)-enantiomer, the selectivity quantified by an enantiomeric ratio (E) value of 7. From a comparison of the apparent kinetic parameter values of QH-ADH and ALDH, it is concluded that ALDH is mainly responsible for the removal of glycidaldehyde in conversions of glycidol catalysed by A. pasteurianus cells. It is shown that the transient observed in rac.-glycidol conversion by whole cells, is indeed (R)-glycidaldehyde. Since both QH-ADH and ALDH are responsible for vinegar production from ethanol by Acetobacters, growth and induction conditions optimal for this process seem also suited to yield cells with high catalytic performance with respect to kinetic resolution of glycidol and prevention of formation of inhibitory concentrations glycidaldehyde.

Direct hydride transfer in the reaction mechanism of quinoprotein alcohol dehydrogenases: a quantum mechanical investigation.

Oxidation of alcohols by direct hydride transfer to the pyrroloquinoline quinone (PQQ) cofactor of quinoprotein alcohol dehydrogenases has been studied using ab initio quantum mechanical methods. Energies and geometries were calculated at the 6-31G(d,p) level of theory. Comparison of the results obtained for PQQ and several derivatives with available structural and spectroscopic data served to judge the feasibility of the calculations. The role of calcium in the enzymatic reaction mechanism has been investigated. Transition state searches have been conducted at the semiempirical and STO-3G(d) level of theory. It is concluded that hydride transfer from the Calpha-position of the substrate alcohol (or aldehyde) directly to the C(5) carbon of PQQ is energetically feasible. Copyright 2001 John Wiley & Sons, Inc. J Comput Chem 22: 1732-1749, 2001

Solvent effect on lipase enantioselectivity. Evidence for the presence of two thermodynamic states.

The enantioselectivity of lipase-catalyzed kinetic resolutions has been measured at various temperatures in binary mixtures of solvents. Varying the solvent composition and temperature had a profound effect on the enantiomeric ratio. The values for delta delta H(R-S)(#) and delta delta S(R-S)(#), calculated from the E values measured at various temperatures, were estimated as a function of the solvent composition. By plotting delta delta H(R-S)(#) versus delta delta S(R-S)(#) as a function of the solvent composition, an extreme was observed. The resulting "hairpin-type" enthalpy-entropy compensation plots can be described by assuming the presence of two thermodynamically distinct physical states, displaying different enantioselectivities, that are in equilibrium with one another. Changing the solvent composition results in a change in the equilibrium constant K(eq) for the two states. The intriguing bell-shaped curves of the enantioselectivity versus solvent composition observed for lipase-catalyzed kinetic resolutions can be described assuming a linear correlation for the logarithm of K(eq) and the solvent composition. Thus, a simulation of the two-state model adequately describes the solvent effects found for lipase-catalyzed kinetic resolutions in binary mixtures of solvents and possibly in series of homologous organic solvents.

Hydrolytic activity in baker's yeast limits the yield of asymmetric 3-oxo ester reduction.

Microbial reductions of ketones hold great potential for the production of enantiopure alcohols, as long as highly selective redox enzymes are not interfered with by competing activities. During reduction of ethyl 3-oxobutanoate by baker's yeast (Saccharomyces cerevisiae) to ethyl (S)-3-hydroxybutanoate, a high enantiomeric excess (> 99%) can be obtained. However, reported yields do not exceed 50-70%. In this article, three main causes are shown to be responsible for these low to moderate yields. These are evaporation of the substrate and product esters, absorption or adsorption of the two esters by the yeast cells and hydrolysis of the two esters by yeast enzymes. The hydrolysis products are further metabolized by the yeast. By reducing the evaporation and absorption losses, the reduction yield can easily be improved to about 85%. Improvement of the efficiency of the reduction and hence the reduction/hydrolysis ratio should lead to a further increase in yield.

Competitive lipase-catalyzed ester hydrolysis and ammoniolysis in organic solvents; equilibrium model of a solid-liquid-vapor system.

Enzymatic ester hydrolysis and ammoniolysis were performed as competitive reactions in methyl isobutyl ketone without a separate aqueous phase. The reaction system contained solid ammonium bicarbonate, which dissolved as water, ammonia, and carbon dioxide. During the reaction an organic liquid phase, a vapor phase, and at least one solid phase are present. The overall equilibrium composition of this multiphase system is a complex function of the reaction equilibria and several phase equilibria. To gain a quantitative understanding of this system a mathematical model was developed and evaluated. The model is based on the mass balances for a closed batch system and straightforward relations for the reaction equilibria and the solubility equilibria of ammonium bicarbonate, the fatty acid ammonium salt, water, ammonia, and carbon dioxide. For butyl butyrate as a model ester and Candida antarctica lipase B as the biocatalyst this equilibrium model describes the experiments satisfactorily. The model predicts that high equilibrium yields of butyric acid can be achieved only in the absence of ammoniolysis or in the presence of a separate water phase. However, high yields of butyramide should be possible if the water concentration is fixed at a low level and a more suited source of ammonia is applied.

Understanding the influence of temperature change and cosolvent addition on conversion rate of enzymatic suspension reactions based on regime analysis.

It is a commonly held belief that enzymatic conversions of substrate in aqueous suspensions can be speeded up by raising the temperature or adding organic solvents to promote dissolution of the substrate. To quantify the impact of such changes, we studied the alpha-chymotrypsin-catalyzed hydrolysis of dimethyl benzylmethylmalonate as a model system. It was found that, upon addition of organic cosolvents, longer process times were actually required, even though the substrate solubility increased severalfold as expected. Upon raising the temperature from 25 degrees C to 37 degrees C, on the other hand, both the substrate solubility, the substrate dissolution rate, and the enzymatic reaction rate increased, leading to shorter process times. A dissolution-reaction model incorporating the kinetics of enzyme deactivation could be developed. A simple relation for the prediction of the overall process time was established by evaluating the time constants for the subprocesses: substrate dissolution; enzymatic conversion; and enzyme deactivation. Using regime analysis, rules of thumb for the optimization of an enzymatic suspension reaction were derived.

Characterization of the Enantioselective Properties of the Quinohemoprotein Alcohol Dehydrogenase of Acetobacter pasteurianus LMG 1635. 1. Different Enantiomeric Ratios of Whole Cells and Purified Enzyme in the Kinetic Resolution of Racemic Glycidol.

Resting cells of Acetobacter pasteurianus LMG 1635 (ATCC 12874) show appreciable enantioselectivity (E=16-18) in the oxidative kinetic resolution of racemic 2,3-epoxy-1-propanol, glycidol. Distinctly lower values (E=7-9) are observed for the ferricyanide-coupled oxidation of glycidol by the isolated quinohemoprotein alcohol dehydrogenase, QH-ADH, which is responsible for the enantiospecific oxidation step in whole cells. The accuracy of E-values from conversion experiments could be verified using complementary methods for the measurement of enantiomeric ratios. Effects of pH, detergent, the use of artificial electron acceptors, and the presence of intermediate aldehydes, could be accounted for. Measurements of E-values at successive stages of the purification showed that the drop in enantioselectivity correlates with the separation of QH-ADH from the cytoplasmic membrane. It is argued that the native arrangement of QH-ADH in the membrane-associated complex favors the higher E-values. The consequences of these findings for the use of whole cells versus purified enzymes in biocatalytic kinetic resolutions of chiral alcohols are discussed.

Homology model of the quinohaemoprotein alcohol dehydrogenase from Comamonas testosteroni.

A molecular model of QH-ADH, the quinohaemoprotein alcohol dehydrogenase from Comamonas testosteroni, has been built by homology modelling. Sequence similarity of N-terminal residues 1-570 with the alpha-subunit of quinoprotein methanol dehydrogenases (MDHs) from Methylophilus methylotrophus W3A1 and Methylobacterium extorquens provided a basis for the design of the PQQ-binding domain of QH-ADH. Minimal sequence similarity with cytochrome c551 from Ectothiorhodospira halophila and cytochrome c5 from Azotobacter vinelandii has been used to model the C-terminal haem c-binding domain, residues 571-677, absent in MDHs. Distance constraints inferred from 19F-NMR relaxation studies of trifluoromethylphenylhydrazine-derivatized PQQ bound to QH-ADH apoenzyme as well as theoretical relations for optimal electron transfer have been employed to position the haem- and PQQ-binding domains relative to each other. The homology model obtained shows overall topological similarity with the crystal structure of cd1-nitrite reductase from Thiosphera pantotropha. The proposed model accounts for the following: (i) the site that is sensitive to in vivo proteolytic attack; (ii) the substrate specificity in comparison with MDHs; (iii) changes of the spectral properties of the haem c upon reconstitution of apo-enzyme with PQQ; (iv) electronic interaction between haem and PQQ; and (v) enantioselectivity in the conversion of a chiral sec alcohol.

Simple conformation space search protocols for the evaluation of enantioselectivity of lipases.

Two computational protocols have been evaluated regarding their ability to reproduce the enthalpic part of lipase enantioselectivity by forcefield potential energy differences (deltaV#R-S). Though the shortcomings of the approach are numerous, good qualitative results have been obtained here and elsewhere. The anticipated improvement of quantitative results by use of a second protocol, which did not impose any atom movement restrictions on the total system, was realized only in part. Seemingly, results depended not only on the design of the computational procedure but also on the enzyme-substrate combination modelled. With Candida antarctica lipase B, results diverged significantly more from an estimated deltadeltaH#R-S than with Rhizomucor miehei lipase and cutinase.

Simple dissolution-reaction model for enzymatic conversion of suspension of solid substrate.

Although reactions in substrate suspension are employed in industry for several bioconversion processes, there appears to be no quantitative model available in the literature to rationalize the optimization of these processes. We present a simple model that incorporates the kinetics of substrate dissolution and a simultaneous enzymatic reaction. The model was tested in the alpha-chymotrypsin-catalyzed hydrolysis of an aqueous suspension of dimethyl benzylmethylmalonate to a homogeneous solution of enantiomerically pure monoester. This reaction occurs in the bulk phase, so catalysis by enzyme absorbed at the solid-liquid interface plays no role. The value of the parameters in the model (i.e., the mass transfer coefficient of substrate dissolution (k(L)), the substrate solubility, and the rate constant for the enzymatic reaction) were determined in separate experiments. Using these parameter values, the model gave a good quantitative prediction of the rate of the overall dissolution-reaction process. When the particle size distribution is known, k(L) may also be calculated instead. The model seems to be applicable also for other poorly soluble substrates, other enzymes, and other solvents. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 433-440, 1997.

Characterization of the gene encoding quinohaemoprotein ethanol dehydrogenase of Comamonas testosteroni.

The gene encoding quinohaemoprotein ethanol dehydrogenase type I (QH-EDHI) from Comamonas testosteroni has been cloned and sequenced. Comparison of the amino acid sequence deduced from this with that determined for the N-terminal amino acid stretch of purified QH-EDHI, suggests that the gene also contains a leader sequence of 31 residues. Based on this information, the molecular mass of the apo-enzyme, i.e. the enzyme without the cofactors pyrroloquinoline quinone (PQQ) and haem c, and without the Ca2+, appears to be 73 200 Da. Alignment of the deduced amino acid sequence to that of other PQQ-containing dehydrogenases showed that good similarity (up to 43% identity) exists with most of them. This also showed that the amino acid residues presumed to be involved in PQQ and Ca2+ binding and in the typical features of structure and catalysis of methanol dehydrogenase, are conserved at the same positions in QH-EDHI. The C-terminal part of the protein, containing the haem c, exhibited some similarity to cytochromes C553 from cyanobacteria and algae. Correct processing of the qhedh gene appeared to occur in Escherichia coli strain JM 109 in which the gene was placed under control of the lac promoter, as judged from a positive reaction with antibodies raised against authentic QH-EDHI, the size of the protein, the presence of haem c in it, and the specific activity value obtained after reconstitution with PQQ. The qhedh gene seems to form part of an operon which is organized in a way different from that of the genes required for methanol oxidation in methylotrophic bacteria.

Thermodynamic and kinetic parameters of lipase-catalyzed ester hydrolysis in biphasic systems with varying organic solvents.

Kinetics of lipase-catalyzed hydrolysis of esters were modeled using reactant activities for aqueous-organic, biphasic systems. By using thermodynamic activities of the substrates in ordinary rate equations, the kinetic parameters were corrected for the contribution of substrate-solvent interactions and a uniform quantification of the substrates for lipase attached to the interface can be achieved. The kinetic parameters, on the basis of their thermodynamic activities, should be constant in different systems, provided that the solvents do not interfere with the binding of the substrates to the enzyme nor affect the catalytic mechanism. Experimental and computational methods on how to obtain the thermodynamic activities of the substrates are presented. Initial rates were determined for Pseudomonas cepacia lipase (PcL)-catalyzed hydrolysis of decyl chloroacetate in dynamic emulsions with various solvents. The thermodynamic equilibrium and corrected kinetic constants for this reaction appeared to be similar in various systems. The kinetics of PcL in an isooctane-aqueous biphasic system could be adequately described with the rate equation for a ping-pong mechanism. The observed inhibitory effect of decanol appeared to be a consequence of this mechanism, allowing the backreaction of the decanol with the chloroacetyl-enzyme complex. The kinetic performance of PcL in systems with toluene, dibutyl ether, and methyl isobutyl ketone could be less well described. The possible causes for this and for the remaining differences in corrected kinetic parameters are discussed.

Identification of topaquinone, as illustrated for pig kidney diamine oxidase and Escherichia coli amine oxidase.

Pig kidney diamine oxidase was purified to homogeneity. The reaction product of the cofactor with p-nitrophenylhydrazine (pNPH) was liberated with pronase treatment and purified. 1H NMR, uv/vis, and electrospray tandem mass spectroscopy revealed it to be a dipeptide with the sequence topaquinone-pNPH and aspartate. No heterogeneity was observed, indicating that no intramolecular cyclization of the quinone moiety occurs in the time span of the isolation and of the measurements. Similar results were obtained with the more widely applicable reagent, phenylhydrazine, and using the aromatic amine oxidase from Escherichia coli. From the amount and ease with which the dipeptide could be isolated, the procedure used here is more convenient than the existing one for the identification of protein-integrated quinone cofactors.

Do organic solvents affect the catalytic properties of lipase? Intrinsic kinetic parameters of lipases in ester hydrolysis and formation in various organic solvents.

When it is assumed that organic solvents do not interfere with the binding process nor with the catalytic mechanism, the contribution of substrate-solvent interactions to enzyme kinetics can be accounted for by just replacing substrate concentrations in the equations by thermodynamic activities. It appears from the transformation that only the affinity parameters (K(m), K(sp)) are affected by this. Thus, in theory, the values of these corrected, intrinsic parameters (K(m) (int), k(sp) (int)) and the maximal rate (V(1)) should be equal for all media. This was tested for hydrolysis, transesterification, and esterification reactions catalyzed by pig pancreas lipase and Pseudomonas cepacia lipase in various organic solvents. Correction was carried out via experimentally determined activity coefficients for the substrates in these solvents or, if not feasible, from values in data bases. However, although the kinetic performances of each enzyme in the solvents became much more similar after correction, differences still remained. Analysis of the enzyme suspensions revealed massive particles, which explains the low activity of enzymes in organic solvents. However, no correlation was found between estimates of the amount of catalytically available enzyme (present at the surface of suspended particles or immobilized on beads) and the maximal rates observed. Moreover, the solvents had similar effects on the intrinsic parameters of suspended and immobilized enzyme. The possible causes for the effects of the solvents on the catalytic performance of the enzymes, remaining after correction for solvent-substrate interactions and the amount of participating enzyme, are discussed with respect to the premises on which the correction method is based. (c) 1995 John Wiley & Sons, Inc.

Characterization of the interaction between PQQ and heme c in the quinohemoprotein ethanol dehydrogenase from Comamonas testosteroni.

Quinohemoprotein ethanol dehydrogenase from Comamonas testosteroni (QH-EDH) contains two cofactors, 2,7,9-tricarboxy-1H-pyrrolo[2,3-f]quinoline-4,5-dione (PQQ) and heme c. Since previous studies on the kinetics of this enzyme suggested that both participate in electron transfer, spectroscopic investigations were performed of the oxidized and reduced holo- and apoenzyme (without PQQ but with heme c) to reveal the nature of the interaction between the two redox centers. From this it appears that the properties of the heme in the enzyme are affected by the presence of PQQ, as judged from the shift of the maxima in the ultraviolet/visible absorption spectra of the heme moiety in both reduced and oxidized QH-EDH and the 60-mV increase of the heme midpoint redox potential caused by PQQ addition. Also 1H-NMR spectroscopy was indicative for interaction since binding of PQQ induced shifts in the resonances of the methyl groups of the porphyrin ring in the oxidized form of the apoenzyme and a shift in the methionine heme ligand resonance of the reduced form of the apoenzyme. On the other hand, resonance Raman spectra of the heme in the different enzyme forms were nearly similar. These results suggest that a major effect of PQQ binding to apo-QH-EDH is a rotation of the methionine ligand of heme c. Since no intermediate 1H-NMR spectra were observed upon titration of apoenzyme with PQQ, apparently no exchange occurs of PQQ between (oxidized) holo- and apoenzyme at the NMR time scale and at that of the experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

Quinohaemoprotein ethanol dehydrogenase from Comamonas testosteroni. Purification, characterization, and reconstitution of the apoenzyme with pyrroloquinoline quinone analogues.

Pyrroloquinoline-quinone(PQQ)-free quinohaemoprotein ethanol dehydrogenase (QH-EDH) apoenzyme was isolated from ethanol-grown Comamonas testosteroni. The purified apoenzyme, showing a single band of 71 kDa on native gel electrophoresis, could be only partially converted into active holoenzyme by addition of PQQ in the presence of calcium ions. In addition to a band with a molecular mass of 71 kDa, additional bands of 51 kDa and 25 kDa were observed with SDS/PAGE. Analysis of the N-terminal sequences of the bands and comparison with the DNA sequence of the gene, suggested that the latter two originate from the former one, due to scission occurring at a specific site between two vicinal residues in the protein chain. The extent of scission appeared to increase during growth of the organism. After addition of PQQ to apoenzyme, holoenzyme and nicked, inactive enzyme could be separated. Holoenzyme prepared in this way was found to contain equimolar amounts of PQQ, Ca2+ and covalently bound haem. EPR spectra of fully oxidized apo-QH-EDH and holo-QH-EDH showed g values typical for low-spin haem c proteins. In partially oxidized holo-QH-EDH an organic radical signal attributed to the semiquinone form of PQQ was observed. Binding of PQQ leads to conformational changes, as reflected by changes of spectral and chromatographic properties. Reconstitution of apoenzyme with PQQ analogues resulted in a decreased activity and enantioselectivity for the oxidation of chiral alcohols. Compared with PQQ, analogues with a large substituent had a lower affinity for the apoenzyme. Results with other analogues indicated that possession of the o-quinone/o-quinol moiety is not essential for binding but it is for activity.

Factors relevant to the production of (R)-(+)-glycidol (2,3-epoxy-1-propanol) from racemic glycidol by enantioselective oxidation with Acetobacter pasteurianus ATCC 12874.

Acetobacter pasteurianus oxidizes glycidol with high activity, comparable to the oxidation of ethanol. The organism has a preference for the S-enantiomer, and the kinetic resolution process obeys a simple relationship, indicating an enantiomeric ratio (E) of 19. The compound is converted into glycidic acid, although a transient accumulation of glycidaldehyde occurs initially. Determination of other parameters revealed a temperature optimum of 50 degrees C, long-term stability (cells in the resting state), and a pH optimum compatible with the chemical stability of glycidol. However, it was also noted that respiration rates decrease at concentrations of glycidol above 1 M. This is most likely caused by substrate inhibition of the glycidol-oxidizing enzyme, the quinohemoprotein ethanol dehydrogenase. Comparison with existing methods for enantiomerically pure glycidol production indicated a number of attractive points for the method described here, although definitive evaluation must await further studies on the long-term stability under process conditions, reusability of the cells, and the mechanism of glycidol inhibition.