The enzymatic reaction-induced configuration change of the prosthetic group PQQ of methanol dehydrogenase.

Methanol dehydrogenase is a heterotetrameric enzyme containing the prosthetic group pyrroloquinoline quinone (PQQ), which catalyzes the oxidation of methanol to formaldehyde. The crystal structure of methanol dehydrogenase from Methylophilus W3A1, previously determined at high resolution, exhibits a non-planar configuration of the PQQ ring system and lends support for a hydride transfer mechanism of the enzymatic reaction catalyzed by the enzyme. To investigate why PQQ is in the C5-reduced form and to better understand the catalytic mechanism of the enzyme, three structures of this enzyme in a new crystal form have been determined at higher resolution. Two of the three crystals were grown in the presence of 1 and 50 mM methanol, respectively, both structures of which show non-planar configurations of the PQQ ring system, confirming the previous conclusion; the other was crystallized in the presence of 50 mM ethanol, the structure of which displays a planar ring system for PQQ. Comparison of these structures reveals that the configuration change of PQQ is induced by the enzymatic reaction. The reaction takes place and the C5-reduced PQQ intermediate is produced when the enzyme co-crystallizes with methanol, but the enzymatic reaction does not take place and the PQQ ring retains a planar configuration of the oxidized orthoquinone form when ethanol instead of methanol is present in the crystallization solution.

Fluorescence lifetimes of tyrosine residues in cytochrome c'' as local probes to study protein unfolding.

Time-resolved fluorescence spectroscopy was used to show that multiple tyrosine residues of a protein can serve as localized probes of structural changes during thermal unfolding. Cytochrome c'' from Methylophilus methylotrophus, which has four tyrosine residues, was chosen as a model protein. The procedure involved, first, the assignment of the experimental decay times to the tyrosine residues, followed by the interpretation of the changes in the decay times and pre-exponential coefficients with temperature. We found that the fluorescence decays of cytochrome c'' are double-exponential from 23 to 80 degrees C, with decay times much shorter than those of the parent compound N-acetyl-tyrosinamide; this quenching was ascribed to dipole-dipole energy transfer from the tyrosine residues to the heme. The tyrosine-heme distances (R) and theoretical decay times, tau(comp), were estimated for each tyrosine residue. The analysis of the simulated decay generated with tau(comp), showed that a double-exponential fit is sufficient to describe the four decay times with two pre-exponential coefficients close to values observed from the experimental decay. Therefore, the decay times at 23 degrees C could be assigned to the individual tyrosine residues as tau(1) to Tyr-10 and Tyr-23 (at 20.3 A) and tau(2) to Tyr-12 and Tyr-115 (at 12-14 A). On the basis of this assignment and MD simulations, the temperature dependence of the decay times and pre-exponential coefficients suggest that upon unfolding, Tyr-12 is displaced from the heme, with loss of the structure of alpha-helix I. Moreover, Tyr-115 remains close to the heme and the structure in this region of the protein is not altered significantly. Altogether the data support the view that the protein core, comprising the heme and the four alpha-helices II to V, is clearly more stable than the remaining region that includes alpha-helix I and the loop between residues 19-27.

[Effect of deuteration on methanol dehydrogenase activity in Methylophilus sp. B-7741]. / Vliianie deiterirovaniia na aktivnost' metanoldegidrogenazy Methylophilus sp. B-7741.

We studied the effect of deuterium oxide present in the medium on the activity of methanol dehydrogenase (EC 1.1.99.8) from methylotrophic bacteria Methylophilus sp. B-7741. Methanol dehydrogenase activity in extracts of the biomass obtained in a highly deuterated medium (2H-enzyme) was 34-47% of enzyme activity in the control biomass, which depended on reaction conditions. The isotopic effects of substrate deuterium (methanol) for 1H-enzyme and 2H-enzyme were 1.37 +/- 0.05 and 1.38 +/- 0.01, respectively. We revealed for the first time the reverse isotopic effect of solvent deuterium in the reaction catalyzed by methanol dehydrogenase (0.80 +/- 0.02 and 0.60 +/- 0.01 for 1H-enzyme and 2H-enzyme, respectively).

Catalytic mechanism of dichloromethane dehalogenase from Methylophilus sp. strain DM11.

The glutathione (GSH)-dependent dichloromethane dehalogenase from Methylophilus sp. strain DM11 catalyzes the dechlorination of CH(2)Cl(2) to formaldehyde via a highly reactive, genotoxic intermediate, S-(chloromethyl)glutathione (GS-CH(2)Cl). The catalytic mechanism of the enzyme toward a series of dihalomethane and monohaloethane substrates suggests that the initial addition of GSH to the alkylhalides is fast and that the rate-limiting step in turnover is the release of either the peptide product or formaldehyde. With the exception of CH(2)ClF, which forms a relatively stable GS-CH(2)F intermediate, the turnover numbers for a series of dihalomethanes fall in a very narrow range (1-3 s(-1)). The pre-steady-state kinetics of the DM11-catalyzed addition of GSH to CH(3)CH(2)Br exhibits a burst of S-(ethyl)-glutathione (k(b) = 96 +/- 56 s(-1)) followed by a steady state with k(cat) = 0.13 +/- 0.01 s(-1). The turnover numbers for CH(3)CH(2)Cl, CH(3)CH(2)Br, and CH(3)CH(2)I are identical, indicating a common rate-limiting step. The turnover numbers of the enzyme with CH(3)CH(2)Br and CH(3)CH(2)I are dependent on viscosity and are very close to the measured off-rate of GSEt. The turnover number with CH(2)I(2) is also dependent on viscosity, suggesting that a diffusive step is rate-limiting with dihaloalkanes as well. The rate constants for solvolysis of CH(3)SCH(2)Cl, a model for GS-CH(2)Cl, range between 1 s(-1) (1:1 dioxane/water) and 64 s(-1) (1:10 dioxane/water). Solvolysis of the S-(halomethyl)glutathione intermediates may also occur in the active site of the enzyme preventing the release of the genotoxic species. Together, the results indicate that dissociation of the GS-CH(2)X or GS-CH(2)OH intermediates from the enzyme may be a relatively rare event.

Sequence variation in dichloromethane dehalogenases/glutathione S-transferases.

Dichloromethane dehalogenase/glutathione S-transferase allows methylotrophic bacteria to grow with dichloromethane (DCM), a predominantly man-made compound. Bacteria growing with DCM by virtue of this enzyme have been readily isolated in the past. So far, the sequence of the dcmA gene encoding DCM dehalogenase has been determined for Methylobacterium dichloromethanicum DM4 and Methylophilus sp. DM11. DCM dehalogenase genes closely related to that of strain DM4 were amplified by PCR and cloned from total DNA from 14 different DCM-degrading strains, enrichment cultures and sludge samples from wastewater treatment plants. In total, eight different sequences encoding seven different protein sequences were obtained. Sequences of different origin were identical in several instances. Sequence variation was limited to base substitutions; strikingly, 16 of the 19 substitutions in the dcmA gene itself encoded amino acids that were different from those of the DM4 sequence. The kinetic parameters k(cat) and K:(m), the pH optimum and the stability of representative DCM dehalogenase variants were investigated, revealing minor differences between the properties of DCM dehalogenases related to that from strain DM4.

[Site-directed mutagenesis of the dichloromethane dehalogenase gene from Methylophilus sp. strain DM11].

In order to investigate the role of different residues of Methylophilus sp. strain DM11 dichloromethane dehalogenase for substrate binding, glutathione affinity, and catalytic activity, site-directed mutagenesis studies of the gene encoding the enzyme were carried out. The conserved tryptophane residue at 103 region was respectively substituted by phenylalanine, valine or asparagine. The conserved arginine residue at 109 region was substituted by leucine. The conserved tryptophane residue at 117 region was respectively substituted by tyrosine or phenylalanine. Six mutant enzymes were produced. Among them three possess lower activities, other three do not possess activity. The properties of the mutant enzyme W117Y are very different from wild-type enzyme.