Johannis A. Duine

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.

Glucose dehydrogenase from Acinetobacter calcoaceticus: a 'quinoprotein'.

Many ~seudoinonas and Acetubacter species are capable of oxidizing aldoses to the corresponding lactone, by NAD(P)-independent dehydrogenases [ 11. One of these enzymes, glucose dehydrogenase (glucose: (acceptor) oxidoreductase, EC 1.1.99.-), has been purified from Acinetobucter ca~coaceticus and it appeared to contain a novel prosthetic group of unknown structure [2]. Here, the properties of the prosthetic groups bf glucose dehydrogenase and methanol dehydrogenase (EC 1 .1.99.X, an enzyme which is present in methylatrophic bacteria, grown on methane or methanol) are compared. Furthermore, a procedure for testing the activity of the prosthetic groups and similar compounds, using the apoenzyme of a glucose dehydrogenase, is described. As the properties are similar, it is concluded that the prosthetic group of glucose dehydrogenase is, like that of methanol dehydrogenase [3-S], a ‘pyrroloquinoline quinone’ (PQQ). The name ‘quinoproteins’ is proposed for this distinct class of dehydrogenases.

Structure and mechanism of soluble quinoprotein glucose dehydrogenase

Soluble glucose dehydrogenase (s‐GDH; EC 1.1.99.17) is a classical quinoprotein which requires the cofactor pyrroloquinoline quinone (PQQ) to oxidize glucose to gluconolactone. The reaction mechanism of PQQ‐dependent enzymes has remained controversial due to the absence of comprehensive structural data. We have determined the X‐ray structure of s‐GDH with the cofactor at 2.2 Å resolution, and of a complex with reduced PQQ and glucose at 1.9 Å resolution. These structures reveal the active site of s‐GDH, and show for the first time how a functionally bound substrate interacts with the cofactor in a PQQ‐dependent enzyme. Twenty years after the discovery of PQQ, our results finally provide conclusive evidence for a reaction mechanism comprising general base‐catalyzed hydride transfer, rather than the generally accepted covalent addition‐elimination mechanism. Thus, PQQ‐dependent enzymes use a mechanism similar to that of nicotinamide‐ and flavin‐dependent oxidoreductases.

Purification and properties of methanol dehydrogenase from Hyphomicrobium X

(1) A method for the isolation of methanol dehydrogenase (alcohol:(acceptor) oxidoreductase, EC 1.1.99.8) from Hyphomicrobium X is decribed. The purified enzyme was resolved by polyacrylamide gel electrophoresis into one main and two minor active bands. Iron and manganese were the only detected metals in the enzyme preparation. (2) The substrate, methanol, was oxidized to formic acid by a stoichiometric amount of artificial electron acceptor. During the reaction, no free formaldehyde could be detected. Other primary alcohols were oxidized to the corresponding aldehydes were a poor substrate or no substrate at all. (3) Some new and efficient one-electron acceptors were found. With these electron acceptors, the enzyme had a high pH optimum and ammonia was still required in the assay system. (4) ESR spectroscopy showed the presence of an enzyme-bound organic free radical. With X-band ESR the signal had a peak-to-peak linewidth of about 0.7 mT. The signal was further resolved by Q-band ESR and the values gparallel = 2.0024 and gperpendicular = 2.0056 were derived. (5) Under denaturing conditions the ESR signal and enzymatic activity disappeared at the same time as fluorescence appeared. Enzymatic activity is not restored when extracted cofactor and apoenzyme are brought together under normal conditions. Some properties of the unusual prosthetic group are presented in a preliminary form.

Structure of quinoprotein methylamine dehydrogenase at 2.25 A resolution.

The three‐dimensional structure of quinoprotein methylamine dehydrogenase from Thiobacillus versutus has been determined at 2.25 A resolution by a combination of multiple isomorphous replacement, phase extension by solvent flattening and partial structure phasing using molecular dynamics refinement. In the resulting map, the polypeptide chain for both subunits could be followed and an X‐ray sequence was established. The tetrameric enzyme, made up of two heavy (H) and two light (L) subunits, is a flat parallellepiped with overall dimensions of approximately 76 x 61 x 45 A. The H subunit, comprising 370 residues, is made up of two distinct segments: the first 31 residues form an extension which embraces one of the L subunits; the remaining residues are found in a disc‐shaped domain. This domain is formed by a circular arrangement of seven topologically identical four‐stranded antiparallel beta‐sheets, with approximately 7‐fold symmetry. In spite of distinct differences, this arrangement is reminiscent of the structure found in influenza virus neuraminidase. The L subunit consists of 121 residues, out of which 53 form a beta‐sheet scaffold of a central three‐stranded antiparallel sheet flanked by two shorter two‐stranded antiparallel sheets. The remaining residues are found in segments of irregular structure. This subunit is stabilized by six disulphide bridges, plus two covalent bridges involving the quinone co‐factor and residues 57 and 107 of this subunit. The active site is located in a channel at the interface region between the H and L subunits, and the electron density in this part of the molecule suggests that the co‐factor of this enzyme is not pyrrolo quinoline quinone (PQQ) itself, but might be instead a precursor of PQQ.

Quinoproteins: a novel class of dehydrogenases

Abstract In the past few years it has become clear that in addition to the well-known NAD(P)-dependent and fluvoprotein dehydrogenases there is another class, the so-called quinoproteins, in which pyrrolo-quinoline quinone (PQQ) is involved as the coenzyme.

Determination of absorption coefficients of purified proteins by conventional ultraviolet spectrophotometry and chromatography combined with multiwavelength detection

The direct, ultraviolet spectrophotometric determination of protein absorption coefficients was found to be more reproducible and accurate when diluting was replaced by chromatography and multiwavelength detection. Four different ultraviolet spectrophotometric methods, described in the literature, were compared by calculating A0.1%280 values from the spectra of 25 proteins, obtained by chromatography. Only two methods, i.e., one based on the absorbance at 210 nm and the other on the absorbance at 205 nm, corrected for the absorbance of aromatic amino acids at that wavelength, were sufficiently accurate to be of potential use for the determination of unknown proteins. It was found, however that with uncorrected A203 values even better results could be achieved. Using 7 well-defined proteins the equation A0.1%280 = 38.69 X A280/A203 - 0.01 was established by linear regression. A0.1%280 values for 14 pure proteins calculated with this equation showed a mean deviation of only 4% from literature values. Since similar deviations were seen with 5 chromophoric and 7 glycoproteins, 3 and 7% respectively, the method may have universal applicability. In the configuration used, only 40 micrograms of a protein is required for the chromatographic determination of its absorption coefficient.

The prosthetic group of methylamine dehydrogenase from Pseudomonas AM1: Evidence for a quinone structure

The g-value and linewidth of ESR spectra of methylamine dehydrogenase (primary-amine:(acceptor) oxidoreductase (deaminating) EC 1.4.99.-) and methanol dehydrogenase (alcohol:(acceptor) oxidoreductase, EC 1.1.99.8) are very similar. This similarity is also reflected in electron-nuclear double resonance (ENDOR) results, the coupling constants of two protons in one enzyme equalling those in the other. The presence of a third proton in the ENDOR spectrum of methylamine dehydrogenase suggests a different structure or a different kind of interaction which can be related to the finding that the resolved ROSTHETIC GROUP IS PROTEIN-BOUND. The bound prosthetic group has a high redox-potential, supporting the conclusion from the ESR and ENDOR results that it is a quinone derivative.

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.

The prosthetic group of methanol dehydrogenase from hyphomicrobium X: Electron spin resonance evidence for a quinone structure

Abstract Partial reduction of the isolated prosthetic group of methanol dehydrogenase yields a free radical with the same characteristics as the one contained in the enzyme. The Electron Spin Resonance spectrum in alkaline aqueous solution displays hyperfine structure and is interpreted in terms of an isotropic g-value, hyperfine coupling constants and nuclear spins. The magnitudes of these parameters indicate that the prosthetic group is a quinone containing two nitrogen atoms.