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Soluble quinoprotein (PQQ-containing) glucose dehydrogenase (sGDH, EC 1.1.99.35) catalyzes the oxidation of β-d-glucose to d-glucono-δ-lactone. Although sGDH has many analytical applications, the relationship between activity and substrate concentration is not well established. Previous steady-state kinetic studies revealed a negative cooperativity effect which has recently been ascribed to subunit interaction. To investigate this conclusion, stopped-flow kinetic experiments were carried out on the reaction in which oxidized enzyme (Eox) was reduced with substrates to Ered. The appearance of Ered is observed to be preceded by formation of an intermediate enzyme form, Int, which is mono-exponentially formed from Eox. However, the rate of conversion of Int into Ered depends hyperbolically on the concentration of substrate (leading to a 35-fold stimulation in the case of glucose). Evidence is provided that substrate not only binds to Eox but also to Int and Ered as well, and that the binding to Int causes the significant stimulation of Int decay. It is proposed that a proton shuffling step is involved in the decay, which is facilitated by binding of substrate to Int. Substituting the PQQ-activating Ca by a Ba ion lowered all reaction rates but did not change the stimulation factor. In summary, the previous proposal that the cooperativity effect of sGDH is due to interaction between its substrate-loaded subunits is incorrect; it is due to substrate-assisted catalysis of the enzyme. Enzymes EC 1.1.99.35 – soluble quinoprotein glucose dehydrogenase.

The enantioselective properties of a large number of enzymes are conveniently characterized by the enantiomeric ratio, E = (kcat/KM)s/(kcat/KM)R. Chen and coworkers [Chen et al., J. Am. Chem. Soc. 104, 7294-9 (1982)] have shown that the value of E can be obtained by measuring the enantiomeric excess, ee, as a function of the degree of conversion, ξ, in kinetic resolutions of a racemic substrate. We noticed that determination of E from ee = f(E, ξ) is not practical when: (a) low amounts of enzyme or substrate are available; (b) analytical procedures for the determination of ee values of substrates or products are cumbersome or inaccurate; (c) complications arise from maturation of reaction mixtures. Direct evaluation of E from the kinetic parameters, on the other hand, requires both enantiomers to be available in enantiomerically pure form. We show that the initial reaction rates for mixtures of enantiomers at fixed substrate concentration under otherwise identical conditions, are related by: (vs, vR and vx represent the initial reaction rate of homochiral S-enantiomer, homochiral R-enantiomer and mixtures with (molar) fraction x of S-enantiomer, respectively). Analytical procedures for the determination of E, vs and vR by this approach are presented. The merits are demonstrated for the lipase-catalysed kinetic resolution of racemic glycidol ester and for the resolution of racemic alcohols by a quinoprotein dehydrogenase and an NAD-dependent dehydrogenase.

Three methods for the determination of the enantiomeric ratio, E, of enantioselective, Michaelis-Menten-type enzymes have been investigated with respect to accuracy, using the method of propagation of standard deviation. In addition, estimations of standard deviations of E were obtained from simulations and representative experiments. The coefficient of variation (γE) achieved in the values derived from the kinetic parameters of the enantiomers (method I) does not depend on the absolute value of E. With initial-rate measurements on mixtures of enantiomers at a fixed concentration of substrates (method II), the accuracy depends on the characteristics of the system, i.e., kinetic parameters, concentration and enantiomeric purity of the substrates, whereas with end-point analysis (method III) the value of E is the crucial factor. Accurate estimates (γE < 0.1) of E can be obtained with method I, and for E < 30 also with method III. As well as the aspect of accuracy, the methods have different intrinsic merits which can also be relevant in making a choice: method I gives information on the reaction mechanism; method II is well suited for screening purposes; method III is attractive for applied studies as it mimics a preparative process. In view of these complementary capabilities, an adequate set of tools seems to be available now for fundamental and applied studies on enantioselectivity.

On storage of methanol dehydrogenase of Hyphomicrobium X as a solution, the electron spin resonance (ESR) spectrum becomes more complicated. Ka-band (35 GHz) spectroscopy reveals that a paramagnetic species with a lower g-vale is formed from pyrrolo-quinoline semiquinone (PQQH) during the ageing process. From the way in which this second paramagnetic species is formed and the absence of an effect of substrate, activator or both on fresh or aged enzyme, it follows that the complex ESR spectrum cannot be used as an argument to support the view that a three-electron reduced form of PQQ exists in methanol dehydrogenase. X-band ESR spectroscopy of an enzyme solution reveals additional peaks at g= 2.03 and g= 2.01 only when the preparation contains O2 and is measured at liquid nitrogen temperature. The results point to the formation of a complex from PQQH and O2, most probably a peroxyl radical which only exists at low temperature. Direct reduction of methanol dehydrogenase, as it is isolated, is possible. The enzyme form thus produced has the same properties as that obtained from reduction of oxidized methanol dehydrogenase with substrate, containing only a low amount of PQQH-but not other free radical species. This substantiates the earlier conclusion that the isolated enzyme is in a half-oxidized form. A view on the mechanism of action of the enzyme is proposed which is quite opposite to the one that was recently advocated [Mincey et al. (1981) Biochemistry, 20, 7502–7509] since former and present results lead to the following conclusions: (a) there is no evidence for the involvement of a three-electron reduced form of PQQ in the catalytic cycle of the enzyme; (b) enzyme molecules containing free radical species are not reduced by substrate; (c) the product is released before the enzyme is oxidized by electron acceptor.

The primary structure of nicotinoprotein alcohol dehydrogenase (ADH) from Amycolatopsis methanolica was determined and used for modelling against known ADH structures, and for evaluation of the coenzyme binding. The results establish the medium-chain dehydrogenase/reductase nature of the nicotinoprotein ADH. Its subunit model and that of the human class Ibeta ADH subunit structure are similar, with mean a carbon deviations of 0.95 A, but they differ in seven loops. Nicotinoprotein ADH occupies a phylogenetic position intermediate between the dimeric and tetrameric ADH families. Two of the differing loops are important for coenzyme binding in the nicotinoprotein model, where one (with a Thr271Arg exchange towards the traditional enzyme) may suggest a slight rotation of the coenzyme adenine ring in the nicotinoprotein, and the other, with an Asn288 insertion, may suggest an extra hydrogen bond to its nicotinamide ribose, favouring stronger binding of the coenzyme. Combined with previous data, this suggests differences in the details of the tight coenzyme binding in different nicotinoproteins, but a common mode for this binding by loop differences.

Previous work has shown that when the bacterium Stenotrophomonas maltophilia is grown on polypropylene glycol, different dye-linked polypropylene glycol dehydrogenase (PPG-DH) activities are induced during growth. Here the purification and characterization of the dehydrogenase activity induced in the stationary phase, and present in the periplasmic space, is described. The homogeneous enzyme preparation obtained consists of a homodimeric protein with a molecular mass of about 123 kDa and an isoelectric point of 5.9. The cofactor of the enzyme appeared to be pyrroloquinoline quinone (PQQ), no heme c was present, and holo-enzyme contained two PQQ molecules per enzyme molecule. In these respects, PPG-DH described here is similar to already known quinoprotein alcohol dehydrogenases, but in other respects, it is different. Therefore, it is suggested that PPG-DH could be a new type of quinoprotein alcohol dehydrogenase. Based on its strong preference for polyols, PPG-DH seems well fitted to carry out the first step in the degradation of PPGs, synthetic polymers containing a variety of hydroxyl groups.

The crystal structure of a quinohemoprotein amine dehydrogenase from Pseudomonas putida has been determined at 1.9-A resolution. The enzyme comprises three non-identical subunits: a four-domain α-subunit that harbors a di-heme cytochromec, a seven-bladed β-propeller β-subunit that provides part of the active site, and a small γ-subunit that contains a novel cross-linked, proteinous quinone cofactor, cysteine tryptophylquinone. More surprisingly, the catalytic γ-subunit contains three additional chemical cross-links that encage the cysteine tryptophylquinone cofactor, involving a cysteine side chain bridged to either an Asp or Glu residue all in a hitherto unknown thioether bonding with a methylene carbon atom of acidic amino acid side chains. Thus, the structure of the 79-residue γ-subunit is quite unusual, containing four internal cross-links in such a short polypeptide chain that would otherwise be difficult to fold into a globular structure.

Penicillium janthinellum F-13 has been isolated in previous work as a fungus tolerating the presence of high concentrations of Al (as high as 100 mM AlCl3). Here its growth rate and yield in three acidic (pH 3.0) media of different composition with varying concentrations of Al are reported. The presence of Al did not affect these parameters, except that the growth yield was somewhat lower in GM (a glucose/peptone/yeast extract-containing medium) with the highest concentration tested (100 mM AlCl3). The amount of Al found in the mycelium was so low that it cannot lead to a significant decrease in the medium for the higher Al concentrations applied. Although citric acid was excreted at growth on GM, and the presence of Al even promoted this, the concentration of this was far too low to diminish (by chelation) the high Al concentrations in the medium to a non-toxic level, i.e. the level (of approx. 1 mM) that is tolerated by low-resistance fungi. At growth on SLBM (a peptone/yeast extract/soil extract-containing medium), a rise in pH occurred. The same was found for SM (a glucose/mineral salts-containing medium), although in this case the picture was more complicated because the initial rise in pH was followed by a lowering due to the excretion of oxalic acid. Although both phenomena can diminish Al toxicity (by decreasing the external concentration of monomeric Al, regarded to be the toxic species), again the decrease is far too low to attain a non-toxic level when high Al concentrations are applied. Therefore, although in principal the metabolic phenomena observed for P. janthinellum F-13 at growth on different media can diminish Al toxicity, the tolerance of this organism for high external Al concentrations must be caused by another mechanism.

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.

Spectroelectrochemical studies were performed on the interaction between Ca2+ and pyrroloquinoline quinone (PQQ) in soluble glucose dehydrogenase (sGDH) and in the free state by applying a mediated continuous-flow column electrolytic spectroelectrochemical technique. The enzyme forms used were holo-sGDH (the holo-form of sGDH from Acinetobacter calcoaceticus) and an incompletely reconstituted form of this, holo-X, in which the PQQ-activating Ca2+ is lacking. The spectroelectrochemical and ESR data clearly demonstrated the generation of the semiquinone radical of PQQ in holo-sGDH and in the free state in the presence of Ca2+. In contrast, in the absence of Ca2+ no semiquinone was observed, either for PQQ in the free state (at pH7.0) or in the enzyme (holo-X). Incorporation of Ca2+ into the active site of holo-X, yielding holo-sGDH, caused not only stabilization of the semiquinone form of PQQ but also a negative shift (of 26.5mV) of the two-electron redox potential, indicating that the effect of Ca2+ is stronger on the oxidized than on the reduced PQQ. Combining these data with the observations on the kinetic and chemical mechanisms, it was concluded that the strong stimulating effect of Ca2+ on the activity of sGDH can be attributed to facilitation of certain kinetic steps, and not to improvement of the thermodynamics of substrate oxidation. The consequences of this conclusion are discussed for the oxidative as well as for the reductive part of the reaction of sGDH.

Until recently, it was generally believed that enzymatic oxidation and reduction requires the participation of either a nicotinamide (NAD(P)+) or a flavin (FAD, FMN), in agreement with the existence of NAD(P)/H–dependent dehydrogenases/reductases and flavoprotein dehydrogenases/reductases/oxidases. However, during the past 20 years, the unraveling of the enzymology of the oxidation and reduction of C1-compounds by bacteria has led to the discovery of many new redox cofactors, some of them discussed here as they have a wider physiological significance than just enabling enzymatic C1-conversions to occur. A good example is the quinone cofactors, encompassing PQQ (2,7,9-tricarboxy-1H-pyrrolo[2,3-f]-quinoline-4,5-dione), TTQ (tryptophyl tryptophanquinone), TPQ (topaquinone), LTQ (lysyl topaquinone), and several others whose structures have still to be elucidated. Another example is mycothiol (1-O-(2′-[N-acetyl-L-cysteinyl]amido-2′-deoxy-α-D-glucopyranosyl)-D-myo-inosoitol), the counterpart of glutathione, once thought to be a universal coenzyme. Because these novel cofactors assist in reactions that can also be catalyzed by already known enzyme “classic cofactor” combinations, and first indications suggest that the chemistry of the reactions is not unique, one may wonder about the evolutionary background for this cofactor diversity. However, as will be illustrated by examples, from a practical point of view the diversity is beneficial, as it has increased the arsenal of enzymes suitable for application. © 2000 John Wiley & Sons, Inc. and The Japan Chemical Journal Forum Chem Rec 1:74–83, 2001

Spectral and kinetic studies were performed on enzyme forms of soluble glucose dehydrogenase of the bacterium Acinetobacter calcoaceticus (sGDH) in which the PQQ-activating Ca(2+) was absent (Holo X) or was replaced with Ba(2+) (Ba-E) or in which PQQ was replaced with an analogue or a derivative called "nitroPQQ" (E-NPQ). Although exhibiting diminished rates, just like sGDH, all enzyme forms were able to oxidize a broad spectrum of aldose sugars, and their reduced forms could be oxidized with the usual artificial electron acceptor. On inspection of the plots for the reductive half-reaction, it appeared that the enzyme forms exhibited a negative cooperativity effect similar to that of sGDH itself under turnover conditions, supporting the view that simultaneous binding of substrate to the two subunits of sGDH causes the effect. Stopped-flow spectroscopy of the reductive half-reaction of Ba-E with glucose showed a fluorescing transient previously observed in the reaction of sGDH with glucose-1-d, whereas no intermediate was detected at all in the reactions of E-NPQ and Holo X. Using hydrazine as a probe, the fluorescing C5 adduct of PQQ and hydrazine was formed in sGDH, Ba-E, and Holo X, but E-NPQ did not react with hydrazine. When this is combined with other properties of E-NPQ and the behavior of enzyme forms containing a PQQ analogue, we concluded that the catalytic potential of the cofactor in the enzyme is not determined by its adduct-forming ability but by whether it is or can be activated with Ca(2+), activation being reflected by the large red shift of the absorption maximum induced by this metal ion when binding to the reduced cofactor in the enzyme. This conclusion, together with the observed deuterium kinetic isotope effect of 7.8 on transient formation in Ba-E, and that already known on transient decay, indicate that the sequential steps in the mechanism of sGDH must be (1) reversible substrate binding, (2) direct transfer of a hydride ion (reversible or irreversible) from the C1 position of the beta-anomer of glucose to the C5 of PQQ, (3) irreversible, rate-determining tautomerization of the fluorescing, C5-reduced PQQ to PQQH(2) and release (or earlier) of the product, D-glucono-delta-lactone, and (4) oxidation of PQQH(2) by an electron acceptor. The PQQ-activating Ca(2+) greatly facilitates the reactions occurring in step 2. His144 may also play a role in this by acting as a general base catalyst, initiating hydride transfer by abstracting a proton from the anomeric OH group of glucose. The validity of the proposed mechanism is discussed for other PQQ-containing dehydrogenases.

The enantioselective properties of copper-containing quinoprotein amine oxidase (EC 1.4.3.6) from Escherichia coli K12 and Klebsiella oxytoca in the kinetic resolution of ( R , S )-1-phenyl-2-aminopropane, amphetamine, have been determined. Determination of the enantiomeric ratio, E =( k cat / K M ) R /( k cat / K M ) S , the ratio of specificity constants for the enantiomeric substrates, can be accomplished in several ways. For practical reasons, we calculated E using non-linear regression analysis of initial rate data obtained at a fixed overall concentration of amphetamine mixtures of chiral composition ranging from 0 to 50% ( R )-(−)-amphetamine [Jongejan et al., Recl. Trav. Chim. Pays-Bas 110 (1990) 247]. It is found that both enzymes catalyze the enantioselective oxidation of amphetamine with E -values of sufficient magnitude ( E ≈15) which may open the possibility for future application of amine oxidase-catalyzed kinetic resolutions of racemic amphetamine. The preference for the ( R )-enantiomer of amphetamine is in agreement with the pro- S specificity that has been observed for the conversion of 2-phenylethylamine. Rationalization of this observation, based on the structure of the E. coli amine oxidase, is discussed.

Quinohaemoprotein alcohol dehydrogenases, QH-ADHs, show appreciable enantioselectivity in the oxidation of certain chiral primary and secondary alcohols. We determined the effect of temperature on the enantiomeric ratio of structurally related enzymes, QH-ADH, Type I, from Comamonas testosteroni and QH-ADH, Type II, from Acetobacrer pasteurianus, in the kinetic resolution of racemic 2, 2-dimethyl-4- (hydroxymethyl)-1, 3-dioxolane (solketal) and 2-butanol, respectively. It appears that entropic contributions to the stabilization of the enantioselectivity-determining transition state play an important role in the enantiopreference. Consequences of these findings for the formulation of models that can be used to summarise the observed enan-tioselectivities are discussed.

Glutathione is not a universal coenzyme for formaldehyde oxidation. MySH (mycothiol, 1-O-(2′-[N-acetyl-L-cysteinyl]amido-2′-deoxy-/propto-D-glucopyranosyl)-D-myo-inositol) is GSH's counterpart as coenzyme in formaldehyde dehydrogenase from certain Gram-positive bacteria. However, formaldehyde dissimilation and detoxification not only proceed via thiol-dependent but also via thiol-independent dehydrogenases. The distinct structures and enzymatic properties of MySH-dependent and GSH-dependent formaldehyde dehydrogenases could provide clues for development of selective drugs against pathogenic Mycobacteria. It is to be expected that other new types of thiol-dependent formaldehyde dehydrogenases will be discovered in the future. Indications exist that the product of thiol-dependent formaldehyde oxidation, the thiol formate ester, is not only hydrolytically converted into thiol and formate but can also be oxidatively converted in some cases by a molybdoprotein aldehyde dehydrogenase into the corresponding carbonate ester, decomposing spontaneously into CO2 and the thiol.

The EPR spectra of three different molybdoprotein aldehyde dehydrogenases, one purified from Comamonas testosteroni and two purified from Amycolatopsis methanolica, showed in their oxidized state a novel type of signal. These three enzymes contain two different [2Fe-2S] centers, one flavin and one molybdopterin cytosine dinucleotide, as cofactors all of which are expected to be EPR silent in the oxidized state. The new EPR signal is isotropic with g = 2.004 both at X-band and Q-band frequencies, consists of six partially resolved lines, and shows Curie temperature behavior suggesting that the signal is due to an organic radical with S = 1/2. The EPR spectra of Comamonas testosteroni aldehyde dehydrogenase obtained after cultivation in media containing 15NH4Cl and/or after substitution of H2O for D2O show the presence of both nitrogen and proton hyperfine interactions. Simulations of the spectra of the four possible isotope combinations yield a single set of hyperfine coupling constants. The electron spin shows hyperfine interaction with a single I = 1 (0.9 mT) ascribed to a N nucleus, with a single I = 1/2 (1.5 mT) ascribed to one nonexchangeable H nucleus, and with two, exchangeable, identical I = 1/2 spins (0.6 mT) ascribed to two identical exchangeable protons. Taken together, the observations and simulations rule out amino acid residues or flavin as the origin of the radical. The values of the various hyperfine coupling constants are consistent with the properties expected for a molybdenum(VI)-trihydropterin radical in which the N5 atom is engaged in two hydrogen-bonding interactions with the protein. The majority of the electron (spin) density of the radical is located at and around the N5 atom and at the proton bound to the C6 atom of the pterin ring. The EPR spectrum of the molybdopterin radical broadens above 65 K and is no longer detectable above 168 K, indicating that it is not magnetically isolated. The line broadening is ascribed to cross-relaxation with a nearby, rapidly relaxing, oxidized [2Fe-2S] center involving its magnetic S = 1 excited state in this process. The amount of radical was apparently not changed by addition of aldehydes or oxidants, but it disappeared upon reduction by sodium dithionite. Therefore, whether the molybdenum(VI) trihydropterin radical as detected here is a functional intermediate in catalysis remains to be investigated further.

The enantioselective properties of lipases in the kinetic resolution of chiral substrates are conveniently expressed as the enantiomeric ratio, E. It has been stated that E is related to the difference of the Gibbs free energy of activation of the enantioselective reaction steps by RT ln E=−ΔΔG#=TΔΔS#−ΔΔH#. From the temperature dependence of E we estimated the enthalpic, ΔΔH#, and entropic, ΔΔS#, contributions. Contrary to earlier suggestions (Aqvist, J. and Warshel, A., 1993. Simulation of enzyme reactions using valence bond force fields and other hybrid quantum/classical approaches. Chem. Rev. 93, 2523–2544.) it is found that the entropic contribution, TΔΔS#, to lipase enantioselectivity at ordinary temperatures is significant. Plots of ΔΔH# versus ΔΔS# for enzyme-catalyzed kinetic resolutions reported in the literature, show a tempting linear correlation of the enthalpic and entropic contributions. On closer inspection, we realized that this is the result of a non-random selection of systems. Hairpin curves are observed for plots of the enthalpic and entropic contributions to lipase-catalyzed enantioselective reactions in water-cosolvent mixtures and in organic media as a function of medium composition. The importance of these findings for the fundamental understanding of enzyme enantioselectivity, the rationalization of solvent effects on the enantiomeric ratio and the prediction of lipase enantioselectivity by molecular modeling techniques is discussed.

Quinohemoprotein amine dehydrogenase (AMDH) was purified and crystallized from the soluble fraction of Pseudomonas putida IFO 15366 grown on n-butylamine medium. AMDH gave a single component in analytical ultracentrifugation showing an intrinsic sedimentation coefficient of 5.8s. AMDH showed a typical absorption spectrum of cytochrome c showing maxima at 554, 522, 420, and 320 nm in the reduced form and one peak at 410 nm, a shoulder at 350 nm, and a broad hill around 530 nm in the oxidized form. The oxidized enzyme was specifically reduced by the addition of amine substrate. AMDH was composed of three different subunits, 60, 40, and 20 kDa, with the total molecular weight of 120,000. Two moles of heme c were detected per mole of AMDH and the 60-kDa subunit was found to be the heme c-carrying subunit. By redox-cycling quinone staining, a positive reaction band corresponding to the 20-kDa subunit was detected after developed by SDS-PAGE, but the 20 kDa band was scarcely stained by conventional protein staining. Only a silver staining method was possible to detect the subunit after the protein was developed by SDS-PAGE. p-Nitrophenylhydrazine-inhibited AMDH was dissociated into subunits and the 20-kDa subunit showed an absorption maximum at 455 nm, indicating Schiff base formation between the carbonyl cofactor in AMDH and the carbonyl reagent. Thus, AMDH is different from nonheme quinoprotein methylamine dehydrogenase and aromatic amine dehydrogenase in many respects. The presence of an azurin-like blue protein was identified and purified from the same cell-free extract of P. putida as AMDH was purified. The blue protein was reduced specifically during AMDH reaction, suggesting that the blue protein is the direct electron acceptor in amine oxidation. The amine oxidation system was reconstituted successfully only by AMDH, the blue protein, and the cytoplasmic membranes of the organism. The function of the 40-kDa subunit is unknown at the moment. The properties of AMDH were compared with other bacterial amine dehydrogenases so far reported.

Prokaryotic mycothiol-dependent formaldehyde dehydrogenase has been structurally characterized by peptide analysis of the 360-residue protein chain and by molecular modelling and functional correlation with the conformational properties of zinc-containing alcohol dehydrogenases. The structure is found to be a divergent medium-chain dehydrogenase/reductase (MDR), at a phylogenetic position intermediate between the cluster of dimeric alcohol dehydrogenases of all classes (including the human forms), and several tetrameric reductases/dehydrogenases. Molecular modelling and functionally important residues suggest a fold of the mycothiol-dependent formaldehyde dehydrogenase related overall to that of MDR alcohol dehydrogenases, with the presence of the catalytic and structural zinc atoms, but otherwise much altered active-site relationships compatible with the different substrate specificity, and an altered loop structure compatible with differences in the quaternary structure. Residues typical of glutathione binding in class-III alcohol dehydrogenase are not present, consistent with that the mycothiol factor is not closely similar to glutathione. The molecular architecture is different from that of the 'constant' alcohol dehydrogenases (of class-III type) and the 'variable' alcohol dehydrogenases (of class-I and class-II types), further supporting the unique structure of mycothiol-dependent formaldehyde dehydrogenase. Borders of internal chain-length differences between this and other MDR enzymes coincide in different combinations, supporting the concept of limited changes in loop regions within this whole family of proteins.

To investigate the mode of binding and the role of Ca2+ in soluble, pyrroloquinoline-quinone (PQQ)-containing glucose dehydrogenase of the bacterium Acinetobacter calcoaceticus (sGDH), the following enzyme species were prepared and their interconversions studied: monomeric apoenzyme (M); monomer with one firmly bound Ca2+ ion (M*); dimer consisting of 2 M* (D); dimer consisting of 2 M and 2 PQQ (Holo-Y); dimer consisting of D with 2 PQQ (Holo-X); fully reconstituted enzyme consisting of Holo-X with two extra Ca2+ ions (Holo) or substitutes for Ca2+ (hybrid Holo-enzymes). D and Holo are very stable enzyme species regarding monomerization and inactivation by chelator, respectively, the bound Ca2+ being locked up in such a way that it is not accessible to chelator. D can be converted into M* by heat treatment and the tightly bound Ca2+ can be removed from M* with chelator, transforming it into M. Reassociation of M* to D occurs spontaneously at 20 degrees C; reassociation of M to D occurs by adding a stoichiometric amount of Ca2+. Synergistic effects were exerted by bound Ca2+ and PQQ, each increasing the affinity of the protein for the other component. Dimerization of M to D occurred with Ca2+, Cd2+, Mn2+, and Sr2+ (in decreasing order of effectiveness), but not with Mg2+, Ba2+, Co2+, Ni2+, Zn2+, or monovalent cations. Conversion of inactive Holo-X into active Holo, was achieved with Ca2+ or metal ions effective in dimerization. Although it is likely that activation of Holo-X involves binding of metal ion to PQQ, the spectral and enzymatic activity differences between normal Holo- and hybrid Holo-enzymes are relatively small. Titration experiments revealed that the two Ca2+ ions required for activation of Holo-X are even more firmly bound than the two required for dimerization of M and anchoring of PQQ. Although the two binding sites related with the dual function of Ca2+ show similar metal ion specificity, they are not identical. The presence of two different sites in sGDH appears to be unique because in other PQQ-containing dehydrogenases, the PQQ-containing subunit has only one site. Given the broad spectrum of bivalent metal ions effective in reconstituting quinoprotein dehydrogenase apoenzymes to active holoenzymes, but the limited spectrum for an individual enzyme, the specificity is not so much determined by PQQ but by the variable metal-ion-binding sites.

Synthesis of enzymes involved in methylamine oxidation via methylamine dehydrogenase (MADH) is encoded by genes present in the mau cluster. Here we describe the sequence of the mauE and mauD genes from Paracoccus denitrificans as well as some properties of mauE and mauD mutants of this organism. The amino acid sequences derived from the mauE and mauD genes showed high similarity with their counterparts in related methylotrophs. Secondary structure analyses of the amino acid sequences predicted that MauE is a membrane protein with five transmembrane-spanning helices and that MauD is a soluble protein with an N-terminal hydrophobic tail. Sequence comparison of MauD proteins from different organisms showed that these proteins have a conserved motif, Cys-Pro-Xaa-Cys, which is similar to a conserved motif found in periplasmic proteins that are involved in the biosynthesis of bacterial periplasmic enzymes containing haem c and/or disulphide bonds. The mauE and mauD mutant strains were unable to grow on methylamine but they grew well on other C1-compounds. These mutants grown under MADH-inducing conditions contained normal levels of the natural electron acceptor amicyanin, but undetectable levels of the beta-subunit and low levels of the alpha-subunit of MADH. It is proposed, therefore, that MauE and MauD are specifically involved in the processing, transport, and/or maturation of the beta-subunit and that the absence of each of these proteins leads to production of a non-functional beta-subunit which becomes rapidly degraded.

The copper quinoprotein amine oxidase from Escherichia coli was derivatized with phenylhydrazines substituted with a F3C group at the ortho, meta, or para position. The derivatization of the topaquinone cofactor was verified by ultraviolet/visible spectroscopy. The reduction (with dithionite) of Cu(II) to Cu(I), which was required to obtain reference samples, was verified by EPR spectroscopy. 19F-NMR spectroscopy was carried out on the derivatized enzyme forms, and the spectra showed the line-broadening effect due to the paramagnetic Cu(II). The distance between the Cu and the mean of the three F positions in the F3C groups was calculated by means of the Solomon-Bloembergen equation for the distance-dependent contribution of Cu(II) to the transversal-relaxation time of the F resonance. Assuming that the F3C-phenylhydrazines in the enzyme are always aligned towards the Cu in the same way, four configurations can be envisaged that should be taken into account to determine the topology of the two cofactors. Based on these configurations, two spatial positions were found where the calculated distances triangulated, each of these positions having a symmetry-related counterpart above or below the topaquinone-phenylhydrazine plane. If it is assumed that the geometric positions of the phenylhydrazine and topaquinone moieties in the adduct remain the same in the derivatized enzymes, a number of minimum distances between the Cu and certain atoms in the topaquinone moiety of the adduct can be calculated (1.52 ± 0.06 nm from the C2-O, 1.30 ± 0.04 nm from the C4-O, and 1.26 ± 0.04 nm from the C5-N). However, one of the configurations yields very similar distances between the Cu and the C2-O and C4-O. Therefore, no conclusions can be made with regard to which OH group is closest to the Cu. By application of the same approach to the 19F-NMR data obtained for porcine-plasma amine oxidase [Williams, T. J. & Falk, M. C. (1986) J. Biol. Chem. 261, 15949–15954] we observed substantial differences between the topologies of the cofactors in the two enzymes. Possible reasons for this are discussed.

Dye-linked alcohol dehydrogenase from Rhodopseudomonas acidophila strain M402, able to oxidize polyethylene glycols, was purified to homogeneity. The monomeric enzyme, having a molecular mass of 72 kDa, contains one PQQ and one haem c per enzyme molecule. In other respects also, the enzyme is very similar to the type I quinohaemoprotein alcohol dehydrogenases known to occur in Comamonas testosteroni, Comamonas acidovorans, and Pseudomonas putida species. However, dissimilarities exist with respect to the isoelectric points and the substrate specificities. On reinvestigating the substrate specificity of the C. testosteroni enzyme, it also appeared to exhibit good activity towards polyethylene glycols. Based on what has been reported for the polyethylene glycol-oxidizing alcohol dehydrogenase of Sphingomonas macrogoltabidus, this enzyme is quite different from that of R. acidophila. Keywords: Polyethylene glycol dehydrogenase activity; Alcohol dehydrogenase; PQQ; Haem c; Rhodopseudomonas acidophila

Methanol-grown Amycolatopsis methanolica NCIB 11946 contains a molybdoprotein dehydrogenase, active with aldehydes and formate esters as substrates and with Wurster's blue as electron acceptor, the so-called formate ester dehydrogenase (FEDH) (van Ophem et al., 1992, Eur. J. Biochem. 206, 519-525). It appears now that another molybdoprotein dehydrogenase is present in this organism. This enzyme, indicated here as dye-linked aldehyde dehydrogenase (DL-AlDH), has the same set of cofactors and converts the same type of substrates but with different specificity, and uses 2,6-dichlorophenol-indophenol as sole artificial electron acceptor for those conversions. The enzymes also differ in their quaternary structure, FEDH having an alpha, beta, gamma and DL-AlDH having an alpha, beta, gamma 2 composition. Furthermore, differences exist with respect to the sizes and the N-terminal amino acid sequences of their subunits, indicating that the enzymes derive from different genes. However, neither their substrate specificity nor their induction pattern give a clear indication for distinct physiological roles. Just like other bacterial molybdoprotein dehydrogenases, DL-AlDH consists of three different subunits (87, 35, and 17 kDa) and contains FAD, molybdopterin-cytosine-dinucleotide cofactor, Fe, and acid-labile sulfide in a molar ratio of 1:1:4:4. Although eukaryotic xanthine oxidase and dehydrogenase differ from these prokaryotic dehydrogenases in size and number of their subunits, certain stretches of amino acid sequences show similarity and the magnetic coupling between the Mo and the [2Fe-2S]-1 cluster in DL-AlDH and bovine milk xanthine oxidase is of the same magnitude. In view of this similarity, the topology of the cofactors in the active site of this type of molybdoproteins might be conserved among enzymes from prokaryotic as well as eukaryotic organisms.

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.

The nonconvertible substrate analogues di-, tri-, and tetramethylammonium are bound with fairly high affinity to oxidized methylamine dehydrogenase (MADHox) from Thiobacillus versutus and induce the same red-shift in the optical absorbance spectrum of MADHox as do the monovalent cations Cs+, Rb+, and NH4+. Like the monovalent cations, trimethylamine also competitively inhibits the reduction of MADHox by methylamine. Rapid-scan experiments show that within the first few milliseconds of the reaction between MADHox and methylamine a red-shifted intermediate is formed as well. Taken together these experiments demonstrate the existence of a common binding site on MADHox for the substrate CH3NH3+, the substrate analogues (CH3)2NH2+, (CH3)3NH+, and (CH3)4N+, and the monovalent cations Cs+, Rb+, and NH4+. Therefore we conclude that, prior to conversion, methylamine is noncovalently bound to MADHox as a cation. The resonance Raman spectra of MADHox in the absence and presence of Cs+, NH4+, and (CH3)3NH+ are very similar, except for the C=O stretching frequencies of the o-quinone carbonyls of the tryptophyltryptophanquinone (TTQ) active center, which show 5-30 cm-1 downshifts. From these Raman results and the X-ray crystal structure, we conclude that the CH3NH3+ binding site is in close proximity to the O6 carbonyl oxygen of the TTQ.

The binding of monovalent cations to methylamine dehydrogenase in the semiquinone state (MADHsq) at a site close to the tryptophan tryptophylquinone (TTQ) active center is demonstrated in experiments which show that the radical EPR signal of MADHsq is considerably broadened in the presence of Cs+, NH4+, and, to a smaller extent, Na+. The cations also stabilize the semiquinone state, as is evident from the increase of the EPR intensity they induce. On the basis of the optical absorbance spectra, two slightly different forms of MADHsq can be discerned. One form, with the main band at 425 nm, is observed at low pH and in the presence of NH4+, whereas the other, with the main band at 429 nm, is observed at high pH and in the presence of Cs+ or Na+. Stopped-flow studies of the oxidation by amicyanin of MADHred via MADHsq to MADHox show a strong stimulation of the first step by monovalent cations. It is shown that it is primarily the actual electron transfer rate, rather than the affinity of MADHred for amicyanin, that is affected by cations. Values for the dissociation constants of the monovalent cations for MADHred, estimated from the kinetic experiments, are higher than those that were previously determined for MADHox, and can be deduced to be higher than those for MADHsq as well. The results are discussed within the context of the electron transfer theory.

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.

A chromosomal fragment containing DNA downstream from mauC was isolated from Paracoccus denitrificans. Sequence analysis of this fragment revealed the presence of four open reading frames, all transcribed in the same direction. The products of the putative genes were found to be highly similar to MauJ, MauG, MauM and MauN of Methylobacterium extorquens AM1. Using these four mau genes, 11 mau genes have been cloned from P. denitrificans to date. The gene order is mauRFBEDACJGMN, which is similar to that in M. extorquens AM1. mauL, present in M. extorquens AM1, seems to be absent in P. denitrificans. MauJ is predicted to be a cytoplasmic protein, and MauG a periplasmic protein. The latter protein contains two putative heme-binding sites, and has some sequence resemblance to the cytochrome c peroxidase from Pseudomonas aeruginosa. MauM is also predicted to be located in the periplasm, but MauN appears to be membrane associated. Both resemble ferredoxin-like proteins and contain four and two motifs, respectively, characteristic for [4Fe-4S] clusters. Inactivation of mauA, mauJ, mauG, mauM and mauN was carried out by introduction of unmarked mutations in the chromosomal copies of these genes. mauA and mauG mutant strains were unable to grow on methylamine. The mauJ mutant strain had an impaired growth rate and showed a lower dye-linked methylamine dehydrogenase (MADH) activity than the parent strain. Mutations in mauM and mauN had no effect on methylamine metabolism. The mauA mutant strain specifically lacked the beta subunit of MADH, but the alpha subunit and amicyanin, the natural electron acceptors of MADH, were still produced. The mauG mutant strain synthesized the alpha and beta subunits of MADH as well as amicyanin. However, no dye-linked MADH activity was found in this mutant strain. In addition, as the wild-type enzyme displays a characteristic fluorescence emission spectrum upon addition of methylamine, this property was lost in the mauG mutant strain. These results clearly show that MauG is essential for the maturation of the beta subunit of MADH, presumably via a step in the biosynthesis of tryptophan tryptophylquinone, the cofactor of MADH. The mau gene cluster mauRFBEDACJGMN was cloned on the broad-host vector pEG400. Transfer of this construct to mutant strains which were unable to grow on methylamine fully restored their ability to grow on this compound. A similar result was achieved for the closely related bacterium Thiosphaera pantotropha, which is unable to utilize methylamine as the sole sources of carbon and energy.

Acinetohacter calcoaceticus LMD 79.41 is a unique bacterium containing a soluble quinoprotein D-glucose dehydrogenase (sGDH) in addition to the membrane-bound quinoprotein D-glucose dehydrogenase (mGDH) which is distributed extensively in Gram-negative bacteria. sGDH has been shown to be a distinct enzyme from mGDH, though both enzymes contain a tightly bound pyrroloquinoline quinone (PQQ) as their prosthetic group. In this study, sGDH was detectable in all strains tested of A. calcoaceticus but not in other Gram-negative bacteria tested, indicating that sGDH can be useful as a taxonomic marker for A. calcoaceticus.The binding process of PQQ with both enzymes was examined by using the apoenzymes purified from a PQQ-deficient mutant strain of A. calcoaceticus. sGDH was able to bind two moles of PQQ in one mole of the homodimer with a fairly high affinity. The binding reaction was much faster at alkaline pH than at acidic pH, and required the presence of some divalent cations such as Cd2+, Ca2+, Sr2+, or Mn...

The kinetic resolution of R,S-glycidyl (R,S-2,3-epoxy-1-propyl) butyrate catalyzed by pig pancreas lipase (PPL) was studied in monophasic and biphasic systems. The course of the resolution at ester concentrations exceeding 0.05 M or in the presence of R,S-glycidol (R,S-2,3-epoxy-1-propanol), could not be described by the equations derived for a one substrate enzyme with a minimal kinetic scheme (Chen et al., 1987). Trivial causes like heterogeneity in activity of the (crude) PPL preparation and equilibrium phenomena due to changing phase ratios could be excluded. An equation based on the kinetic mechanism of hydrolases, in which the acyl-enzyme intermediate is allowed to react with water as well as with the produced alcohol (quantified by the selectivity constant, α), was evaluated. All initial rate and conversion data could be adequately fitted with this equation, not only for PPL in the monophasic (free in solution) but also in the biphasic (adsorbed to the interface) systems where it exhibited better a...

Transesterification of vinyl acetate with racemic glycidol (R,S-2,3-epoxy-1-propanol) by pig pancreas lipase (PPL) was studied in hexane, diisopropylether, tetrachloromethane, and 2-butanone. Correction for substrate-solvent interactions was carried out by using thermodynamic activities of the substrates in the equations. Data from initial rate measurements could be fitted with a Ping Pong Bi Bi model, taking competitive inhibition by glycidol into account. Although plotting of the rates against thermodynamic activities resulted in similar curves for the various solvents, significant variation of the intrinsic parameters still remained. Similarity of the kinetic parameter values increased, however, when competitive inhibition by the solvents was taken into account, suggesting that simple interaction of the solvents with the active site occurs rather than exertion of specific effects on the catalytic properties of the enzyme. It appeared that the enantiomeric ratio, E, and the selectivity factor, α (the ch...

Initial rate studies were performed on the oxidation of (racemic) alcohols as well as aldehydes by quinohaemoprotein ethanol dehydrogenase, type 1, from Comamonas testosteroni with potassium ferricyanide as electron acceptor. The data could be fitted with an equation derived for a mechanism (hexa-uni ping-pong) in which alcohols are oxidized to the corresponding carboxylic acids and the intermediate aldehyde becomes released from the enzyme. However, for some substrates it was necessary to assume that they exert uncompetitive inhibition. The same model was used to fit the data of conversion processes. Reversible inactivation of the enzyme takes place during the conversion, the extent being inversely proportional to the concentration of ferricyanide present at the start. From the values of the kinetic parameters obtained for (R)- and (S)-solketal [2,2-dimethyl-4-(hydroxymethyl)-1,3-dioxolane] and their corresponding aldehydes, it appeared that the second step in (S)-solketal conversion is much faster than the first one and that opposite enantiomeric preferences exist for the alcohol and the aldehyde substrates. Since the initial rate measurements as well as the progress curve analysis gave similar kinetic parameter values and product analysis revealed intermediates in the amounts predicted, it is concluded that the kinetic and enantioselective behaviour of the enzyme is adequately described by the model presented here. Finally, the results indicate that both kinetic approaches should be used in conversions with consecutive reactions since they provide complementary information.

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°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.

Expression of methylamine dehydrogenase in Paracoccus denitrificans and its concomitant ability to grow on methylamine is regulated by a substrate-induction mechanism as well as by a catabolite-repression-like mechanism. Methylamine dehydrogenase is synthesized in cells growing on either methylamine or ethylamine, but not during growth on succinate, methanol or choline as sole sources of carbon and energy. The synthesis of methylamine dehydrogenase is repressed when succinate is added to the growth medium in addition to methylamine. Repression is not observed when the growth medium contains methylamine and either choline or methanol. Induction of the mau genes encoding methylamine dehydrogenase is under control of the mauR gene. This regulatory gene is located directly in front of, but with the transcription direction opposite to that of, the structural genes in the mau cluster. The mauR gene encodes a LysR-type transcriptional activator. Inactivation of the gene results in loss of the ability to synthesize methylamine dehydrogenase and amicyanin, and loss of the ability to grow on methylamine. The mutation is completely restored when the mauR gene is supplied in trans. The first gene of the cluster of mau genes that is under control of MauR is mauF, which encodes a putative membrane-embedded protein. Inactivation of the gene results in the inability of cells to grow on methylamine. Downstream from mauF and in the same transcription direction, mauB is located. This gene encodes the large subunit of methylamine dehydrogenase.

All strains of Comamonas testosteroni investigated here, produced quinohaemoprotein ethanol dehydrogenase (QH-EDH) when grown on ethanol or butanol, but one strain of C. acidovorans and of C. terrigena did not. Hybridization experiments showed that the gene for QH-EDH is absent in the latter two strains. Induction and properties of the QH-EDHs seem to be similar: all C. testosteroni strains produced the enzyme in its apo-form [without pyrroloquinoline quinone (PQQ)] and the levels were higher at growth at low temperature; preference for the R-enentiomer and similar selectivity was shown in the oxidation of solketal (2,2-dimethyl-1,3-dioxolane-4-methanol) by cells (supplemented with PQQ); the fragment of the qhedh gene gave high hybridization with the DNA of the C. testosteroni strains. Experiments with C. testosteroni LMD 26.36 revealed that the organism is well suited for production of (S)-solketal: it shows an adequate enantioselectivity (E value of 49) for the oxidation of racemic solketal; the conversion rate of (R)-solketal is only 3.5 times lower than that of ethanol; the optimal pH for conversion (7.6) is in a region where solketal has sufficient chemical stability; separation of the remaining (S)-solketal from the acid formed is simple; induction of QH-EDH, the sole enzyme responsible for the oxidation of (R)-solketal, occurs during growth on ethanol or butanol so that the presence of solketal (inhibitory for growth) is not required; production of active cells and the conversion step can be integrated into one process, provided that PQQ and solketal addition occur at the appropriate moment; the conversion seems environmentally feasible. However, since high concentrations of solketal inhibit respiration via QH-EDH, further investigations on the mechanism of inhibition and the stability of the enzyme might be rewarding as it could lead to application of higher substrate concentrations with consequently lower down-stream processing costs.

Dye-linked formaldehyde dehydrogenase from methylamine-grown Hyphomicrobium zavarzinii ZV 580, a tetramer of M(r) 210,000 with subunits of M(r) 54,000, was purified to homogeneity in five steps with 10% yield. The enzyme shows optimal affinity for, and activity with, formaldehyde (Km 67 microM) compared with other aldehydes. Pyridoxal phosphate, pyrroloquinoline quinone and other cofactors that would give the enzyme a distinctive absorption spectrum are absent. Slight changes are observed in the spectrum at 300-550 nm on oxidation of the enzyme with Wurster's Blue (WB) and reduction with formaldehyde. Titration of the native reduced enzyme with WB accounts for 2 mol of electrons per mol of tetrameric enzyme. The circumstantial evidence supporting the presence of a redox-active quinone cofactor bound to the polypeptide chain comprises a signal at g = 2.0049 in the X-band e.p.r. spectrum of the enzyme oxidized with WB, which disappears on reduction with formaldehyde, and a positive reaction of the native as well as the denatured and dialysed enzyme in the redox-cycling assay with glycinate and NitroBlue Tetrazolium (quinone staining). The oxidized enzyme is inhibited by equimolar amounts of phenylhydrazine, which is also a reductant. Hydrazone formation was absent with completely inhibited enzyme, according to photometric evidence. Likewise, the glycinate-dependent reduction of NitroBlue Tetrazolium was not affected by the inhibitor. It is concluded that an oxidation product of the hydrazine is the actual inhibitor which reacts with an amino acid residue of the active site rather than with the prospective quinone cofactor.

Quinohaemoprotein alcohol dehydrogenase from Comamonas testosteroni was immobilized on polypyrrole-coated track-etch and microporous membranes. On the track-etch membrane, 3.4 to 4.8 × 10−3 Units of enzyme/cm2 was immobilized whilst on the microporous membrane 0.05 U/cm2 was immobilized. The track-etch membrane was then used in electrochemical studies using ferricyanide as a redox mediator giving a maximum catalytic current of 0.022 mA/cm2 membrane with 1-pentanol as the substrate. The kinetic parameters (Km and Vmax) of the immobilized enzyme are of the same order of magnitude as those of the free enzyme.

Several purified or commercially available alcohol oxidoreductases of different kinds were tested for their ability to convert the racemic, glycerol-based C3-synthons glycidol (2,3-epoxy-1-propanol) and solketal (2,2-dimethyl-4-(hydroxymethyl)-1,3-dioxolane), with adequate activity and enantioselectivity. Quinohaemo-protein alcohol dehydrogenases (enzymes containing haem c as well as pyrroloquinoline quinone (PQQ) as cofactors) appeared to be excellently suited for such use. The bacteria from which the enzymes were purified had the same enantiomer preference and had an efficient respiratory chain for reoxidation of these dehydrogenases. In some cases, however, whole cells gave a lower enantiomer ratio (E) than the pure enzyme. NAD-dependent alcohol dehydrogenases also are present in these bacteria, but their presence may not explain the lower ratio because they oxidized the C3-synthons little if at all. It seems, therefore, that different kinetic mechanisms are responsible for the discrepancy between the ...

Bacteria contain alcohol dehydrogenases (ADH's) which either occur in the cytosol and which interact with the NAD(P) pool or are directly coupled to the respiratory chain and can be assayed i n v i t ro with artificial electron acceptors, i.e. by decoloration o f dyes. During ihe past ten years, several of these dye-linked ADH's have been characterized and shown to be quinoproteins, i.e. enzymes with a quinone cofactor (in most cases this is pyrroloquinoline quinone, PQQ). These enzymes were investigated here for kinetic resolution of the C -synthons solketal ( 2,2-dimethyl-4-( hydroxymethyl) -1,J-dicxslane) and glycidol (2,3-epoxy-l-propanol). It appeared that quinohaemoprotein ADH's are excellently suited for this 4ince this type of enzymes has adequate substrate diversity, catalytic activity and resolving power, i n v i t r o as well as i n vivo.

Accurate and reliable methods are presented for the determination of enantiomeric excess values of glycidol (2,3-epoxy-1-propanol), glycidyl esters, solketal [2,2-dimethyl-4-(hydroxymethyl)-1,3-dioxolane], homologous 1,3-dioxolane alcohols and substituted primary propanols in biological samples. One method consists of derivatization of the samples with 2,3,4,6-tetra-O-acetyl-β-glucopyranosyl isothiocyanate, followed by separation of the resulting diastereomers on a non-chiral reversed-phase (C18) HPLC column. The second method uses direct injection of (aqueous) samples on to capillary GC columns coated with chiral stationary phases (2,3,6-tri-O-methyl-β- or a 2,3,6-tri-O-trifluoroacetyl-λ-cyclodextrin). The advantages and disadvantages of both methods are discussed.

Extracts of Gram-positive bacteria like Rhodococcus rhodochrous, Rhodococcus erythropolis and Amycolatopsis methanolica, but not those of several Gram-negative ones, showed dehydrogenase activity for ethanol as well as for methanol when 4-nitroso-N, N-dimethylaniline (NDMA) was used as electron acceptor. Chromatography of extracts of the first two organisms revealed one activity for both substrates, that of A. methanolica two activities, one of which is able to oxidize methanol and has been purified (Bystrykh, L. V., Govorukhina, N. I., van Ophem, P. W., Hektor, H. J., Dijkhuizen, L. and Duine, J. A., unpublished results). The other, indicated as NDMA-dependent alcohol dehydrogenase (NDMA-ADH), was purified to homogeneity. It is a trimeric enzyme consisting of subunits of 39 kDa and one firmly bound NAD as cofactor. Although NDMA-ADH shows structural similarity with the long-chain, zinc-containing, NAD(P)-dependent alcohol dehydrogenases with respect to the N-terminal sequence up to residue 41 (56% identity with horse liver alcohol dehydrogenase), the enzymes are catalytically different since NDMA-ADH is unable to use NAD(P)(H) as a coenzyme and NAD(P)-dependent alcohol dehydrogenases are inactive with NDMA (in the absence of NAD). Comparison of the NDMA-ADH properties with those of the methanol-oxidizing enzyme of A. methanolica, Mycobacterium gastri and Bacillus methanolica C1, and formaldehyde dismutase of Pseudomonas putida F61 revealed large differences in structural as well as catalytic properties, in spite of the fact that all are nicotinoproteins [enzymes which have bound NAD(P) as a cofactor]. It is concluded, therefore, that NDMA-ADH is a novel type of nicotinoprotein alcohol dehydrogenase.

Glucose electrodes were prepared by wiring quinoprotein glucose dehydrogenase, GDH (EC 1.1.99.17) to glassy carbon with an osmium complex containing redox-conducting epoxy network. Their current density at 70 mM glucose concentration reached 1.8 mA cm -2 when 15 μg cm -2 of the enzyme having an activity of 250 units mg -1 was applied to the electrode. Under the same conditions, electrodes made with glucose oxidase (GOX) of similar activity (250 units mg -1 ) had a maximum current density of 0.66 mA cm -2 . The maximum current density was reached with 8% GDH in the redox polymer film

Quinohaemoprotein alcohol dehydrogenase from Comamonas testosteroni is a functional electron-transfer protein containing both a haem c and a pyrroloquinoline quinone cofactor. The enzyme has been crystallized at 277 K using polyethylene glycol 6000 as precipitant. The crystals belong to space group C2, with unit-cell parameters a = 98.1, b = 74.3, c = 92.2 A, beta = 105.9 degrees. A native data set with a resolution of 2.44 A resolution has been collected. The approximate orientation of the haem group with respect to the unit-cell axes has been determined from the optical properties of the crystals.

Bacteria contain alcohol dehydrogenases (ADH's) which either occur in the cytosol and which interact with the NAD(P) pool or are directly coupled to the respiratory chain and can be assayed i n v i t ro with artificial electron acceptors, i.e. by decoloration o f dyes. During ihe past ten years, several of these dye-linked ADH's have been characterized and shown to be quinoproteins, i.e. enzymes with a quinone cofactor (in most cases this is pyrroloquinoline quinone, PQQ). These enzymes were investigated here for kinetic resolution of the C -synthons solketal ( 2,2-dimethyl-4-( hydroxymethyl) -1,J-dicxslane) and glycidol (2,3-epoxy-l-propanol). It appeared that quinohaemoprotein ADH's are excellently suited for this 4ince this type of enzymes has adequate substrate diversity, catalytic activity and resolving power, i n v i t r o as well as i n vivo.

Publisher Summary Pyrroloquinoline quinone (PQQ, 2,7,9-tricarboxy-1 H -pyrrolo[2,3- f ]quinoline- 4,5-dione) is one of the quinone cofactors functioning in the so-called quinoprotein enzymes. This chapter discusses the isolation, preparation, and assay of PQQ. PQQ functions only as a cofactor in bacterial dehydrogenases catalyzing the oxidation of alcohols or sugars. Covalently bound quinone cofactors are topaquinone and tryptophyltryptophan quinone, which derive from oxidized specific tyrosine and tryptophan residues in the protein chain of the enzymes to which they belong, respectively. The chapter describes the synthesis of several substituted PQQs (with alkyl groups at the N-1, C-3, or C-8 position), of 6-deaza-PQQ, and of decarboxy- PQQs. In addition, the o -quinone moiety of PQQ is modified by reduction or derivatization. The biological activity of some of these analogs is investigated. PQQ, a compound with three carboxylic acid groups, is water soluble when these groups are deprotonated. In principle, anion-exchange chromatography is feasible to detect and determine the compound. A method has been developed in which the PQQ in the sample and added labeled PQQ are chemically modified and the product is investigated by gas chromatography–mass spectrometry (GC–MS). The levels found with this assay in plant materials are orders of magnitude lower than those found with the redox-cycling assay.

Summary: Cytoplasmic membranes of Acinetobacter calcoaceticus cells, cultured under acetate-limiting conditions, contain cytochrome b554 and a cytochrome o-containing oxidase. Both have been purified to homogeneity and characterized. Cytochrome b554 is a monomeric protein (molecular mass 70 kDa) with a midpoint potential of +100 mV (in the membrane it has most probably a value of +50 mV). The cytochrome o-containing oxidase seems to be an αβγδ protein since the molecular mass of the native protein was estimated to be 150 kDa and the molecular masses of the subunits, determined by SDS-polyacrylamide-gel electrophoresis, are 55, 41, 33 and 17 kDa. Redox spectroscopy of the purified complex shows the presence of a cytochrome b555/563 having a midpoint potential of approximately +160 mV (both in purified form and in the membrane). CO difference spectroscopy shows the presence of a second b-type cytochrome, viz. cytochrome, o. Cytoplasmic membranes of A. calcoaceticus cells grown under oxygen-limiting conditions also show the presence of the cytochrome b554 and the cytochrome o-containing oxidase. In addition a protein has been solubilized with the spectral characteristics of a cytochrome d-containing oxidase. The cytochrome o- and d-containing oxidases appear to be similar to those reported for Escherichia coli and Proteus mirabilis. On the other hand, cytochrome b554 has no counterpart in these organisms since the cytochrome b556 described for E. coli is quite dissimilar.