J. A. Jongejan

Bovine serum amine oxidase: a mammalian enzyme having covalently bound PQQ as prosthetic group

In addition to the metal ion, copper‐containing amine oxidases possess an organic prosthetic group, the nature of which has long been controversial. We show here that in the case of bovine plasma amine oxidase, this second prosthetic group is covalently bound pyrroloquinoline quinone (PQQ). Until now the coenzyme PQQ has been found in several bacterial dehydrogenases. Thus the finding reported here is the first example of a quinoprotein oxidoreductase discovered in a eukaryotic organism.

Hydrazone formation of 2,4-dinitrophenylhydrazine with pyrroloquinoline quinone in porcine kidney diamine oxidase

Homogeneous diamine oxidase (EC 1.4.3.6) from porcine kidney was treated with the inhibitor 2,4‐dinitro‐phenylhydrazine (DNPH). The coloured compounds formed were detached with pronase and purified to homogeneity. When the reaction with DNPH was conducted under an O2 atmosphere, the product (obtained in a yield of 55%) was the C(5)‐hydrazone of pyrroloquinoline quinone (PQQ) and DNPH, as revealed by its Chromatographie behaviour, absorption spectrum and 1H‐NMR spectrum. Only 6% of this hydrazone was formed under air, the main product isolated being an unidentified reaction product of DNPH with the enzyme. Porcine kidney diamine oxidase is the second mammalian enzyme shown to have PQQ as its prosthetic group. In view of the requirements for hydrazone formation with DNPH, it is incorrect to assume that inhibition of this type of enzymes with common hydrazines is simply due to blocking of the carbonyl group of its cofactor.

Reductions of 3-oxo esters by baker's yeast : Current status

The objective of the current review is to present a mechanism and process engineering approach of stereospecific reductions of 3-oxo esters by bakers yeast. The stereospecific outcome of a reduction by bakers yeast depends on the kind of 3-oxo ester reductases involved and their specific activity. Various competing 3-oxo ester reductases are present in a yeast cell. An important aspect for efficient biotransformations with whole cells is the regeneration of NADH and NADPH cofactors. Use of different electron donors leads to the involvement of different metabolic routes influencing the reduction process. Optimization of the process conditions such as aeration, immobilization of cells, use of additives, or use of two phases, will enhance re-use of bakers yeast, yield, stereospecific outcome and scale up. Since the genome of bakers yeast is known, genetic engineering will soon increase the possibilities of stereoselective reductions.

Phenylhydrazine as probe for cofactor identification in amine oxidoreductases Evidence for PQQ as the cofactor in methylamine dehydrogenase

Homogeneous methylamine dehydrogenase (primary‐amine:(acceptor) oxidoreductase (deaminating), EC 1.4.99.3, MADH) from the bacterium Thiobacillus versutus was treated with the inhibitor phenylhydrazine (PH). Derivatization of the cofactor in MADH took place in a fast reaction to give compound I. A different product, compound II, was formed in a slow reaction at high O2 concentrations. The compounds I and II could be removed from the protein by proteolysis with pronase and purified to homogeneity. Products showing identical absorption spectra and chromatographic behaviour were isolated from the reaction mixture after incubatng pyrroloquinoline quinone (PQQ) with PH. Upon dissolving in dimethyl sulphoxide, both the enzyme‐derived as well as the model‐system‐derived compounds I and II were nearly quantitatively transformed into PQQ. The conclusion is, therefore, that MADH from T. versutus contains covalently bound PQQ, removable from the protein with pronase, and not a structural analogue of this cofactor without the carboxylic acid groups, as was recently proposed for MADH from Bacterium W3A1 [(1986) Biochem. Biophys. Res. Commun. 141, 562–568]. The properties of compounds I and II suggest that they are the ‘azo adduct’ and the ‘hydrazone adduct’ of PH and PQQ at the C(5)‐position, respectively. The finding that the reaction of a hydrazine with PQQ can lead to two different products, in enzymes as well as in a model system, has important implications for the interpretation of recent comparative studies aimed at detection of PQQ in amine oxidoreductases with Raman spectroscopy.

Detection and determination of pyrroloquinoline quinone, the coenzyme of quinoproteins.

A convenient determination of pyrroloquinoline quinone (PQQ) in extracts of purified enzymes is possible with ion-pair chromatography on a HPLC reverse-phase column and with uv detection. However, when culture supernatants have to be analyzed, a fluorescence detection system is more appropriate. Proof for the presence of PQQ can be obtained by treating such a sample with butyraldehyde, which converts the coenzyme into a stable adduct having a suitable retention time in the system. The sensitivity and selectivity of the analysis can be further enhanced by reducing the sample with NaBH4, which produces the dihydrodiol form of the coenzyme (PQQH4) and oxidizing PQQH4 with NaIO4 to a strongly fluorescing compound. A procedure is described for the easy preparation of an apoenzyme from the quinoprotein glucose dehydrogenase of Pseudomonas aeruginosa strains. With this biological test system, very low amounts of PQQ can be detected. However, when PQQ is present in the form of adducts, the analysis has to be performed via reduction to PQQH4, oxidation with NaIO4, and HPLC.

Dopamine β-hydroxylase from bovine adrenal medulla contains covalently-bound pyrroloquinoline quinone

Treatment of homogeneous dopamine β‐hydroxylase (DBH) preparations from bovine adrenals with the inhibitor phenylhydrazine (PH) changed the structureless absorption spectrum of DBH into spectra with a maximum at 350 nm. A product with this absorption spectrum could be detached with pronase, enabling its isolation. It appeared to be the C(5) hydrazone of pyrroloquinoline quinone (PQQ) and PH, as judged from its properties and the fact that it could be transformed into PQQ itself. From the yield obtained a ratio of 0.85 PQQ per enzyme subunit was calculated. In contrast to copper‐quinoprotein amine oxidases (EC 1.4.3.6), hydrazone formation in DBH did not require saturation of the mixture with O2. DBH is the first copper‐quinoprotein hydroxylase found so far. The implications of this finding for the current views on mechanism of action and inhibition by hydrazines are discussed. The success of the recently developed ‘hydrazine method’ [(1987) FEBS Lett. 221, 299‐304] for all different types of amine oxidoreductases, suggest that the method could also be applied to other enzymes for which hydrazines are inhibitors and where the identity of the cofactors has not been established or the presence of PQQ is suspected.

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.

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.

Pyrroloquinoline Quinone: A Novel Cofactor

Publisher Summary This chapter discusses the properties, distribution, and biosynthesis of pyrroloquinoline quinine (PQQ). PQQ is an essential cofactor for several key enzymes in physiological processes in mammals. Development of inhibitors specifically directed to PQQ might reveal the role of PQQ and quinoproteins, but also that of vitamin B, and enzymes depending on it. This could also be afforded by developing inhibitors specifically blocking the biosynthesis of PQQ or feeding animals a PQQ-free diet. Some analytical procedures are developed to determine PQQ and its derivatives unambiguously in biological materials. The systematic procedures for cofactor identification will stimulate progress on cofactor enzymology. PQQ is detected in several metalloenzymes, which implies that it may also have been overlooked in enzymes already believed to or known to utilize recognized cofactors. The finding of PQQ implies evolutionary variation in the apoprotein of an enzyme and also in its cofactor.

Lipase Mediated Resolution of γ-Branched Chain Fatty Acid Methyl Esters

Kinetic resolution of the branched chain fatty acid (BCFA) esters 4-methylhexanoic acid methyl ester (4) and 4-methyloctanoic acid methyl ester (5) was investigated using a series of hydrolases as catalysts. In the transesterification of these methyl esters to their butyl esters, two enzymes showed good conversion and a moderate enantiomeric ratio (E). In the transesterification of 4, an E of 2 was obtained for the reaction catalysed by Rhizomucor miehei lipase, whereas Candida antarctica lipase B (CALB) showed an E of 5. In the conversion of 5 to the butyl ester, Rhizomucor miehei lipase was unselective whereas CALB gave an E of 8. Apparently, changing from an ethyl group to a butyl group at the chiral centre leads to an improved chiral recognition by CALB. The lipases displayed complementary enantiomeric preference. Rhizomucor miehei lipase favours the S-enantiomer of 4 while CALB preferentially transforms the R-cnantiomer of both substrates. Molecular modelling studies supported the measured stereochem...