Properties of p-cresol methylhydroxylase flavoprotein overproduced by Escherichia coli.

The alpha(2)beta(2) flavocytochrome p-cresol methylhydroxylase (PCMH) from Pseudomonas putida is composed of a flavoprotein homodimer (alpha(2) or PchF(2); M(r) = 119 kDa) with a cytochrome monomer (beta, PchC; M(r) = 9.3 kDa) bound to each PchF subunit. Escherichia coli BL21(DE3) has been transformed with a vector for expression of the pchF gene, and PchF is overproduced by this strain as the homodimer. During purification, it was recognized that some PchF had FAD bound, while the remainder was FAD-free. However, unlike PchF obtained from PCMH purified from P. putida, FAD was bound noncovalently. The FAD was conveniently removed from purified E. coli-expressed PchF by hydroxyapatite chromatography. Fluorescence quenching titration indicated that the affinity of apo-PchF for FAD was sufficiently high to prevent the determination of the dissociation constant. It was found that p-cresol was virtually incapable of reducing PchF with noncovalently bound FAD (PchF(NC)), whereas 4-hydroxybenzyl alcohol, the intermediate product of p-cresol oxidation by PCMH, reduced PchF(NC) fairly quickly. In contrast, p-cresol rapidly reduced PchF with covalently bound FAD (PchF(C)), but, unlike intact PCMH, which consumed 4 electron equiv/mol when titrated with p-cresol (2 electrons from p-cresol and 2 from 4-hydroxybenzyl alcohol), PchF(C) accepted only 2 electron equiv/mol. This is explained by extremely slow release of 4-hydroxybenzyl alcohol from reduced PchF(C). 4-Hydroxybenzyl alcohol rapidly reduced PchF(C), producing 4-hydroxybenzaldehyde. It was demonstrated that p-cresol has a charge-transfer interaction with FAD when bound to oxidized PchF(NC), whereas 4-bromophenol (a substrate analogue) and 4-hydroxybenzaldehyde have charge-transfer interactions with FAD when bound to either PchF(C) or PchF(NC). This is the first example of a "wild-type" flavoprotein, which normally has covalently bound flavin, to bind flavin noncovalently in a stable, redox-active manner.

Crystallographic and spectroscopic studies of native, aminoquinol, and monovalent cation-bound forms of methylamine dehydrogenase from Methylobacterium extorquens AM1.

Various monovalent cations influence the enzymatic activity and the spectroscopic properties of methylamine dehydrogenase (MADH). Here, we report the structure determination of this tryptophan tryptophylquinone-containing enzyme from Methylobacterium extorquens AM1 by high resolution x-ray crystallography (1.75 A). This first MADH crystal structure at low ionic strength is compared with the high resolution structure of the related MADH from Paracoccus denitrificans recently reported. We also describe the first structures (at 1.95 to 2.15 A resolution) of an MADH in the substrate-reduced form and in the presence of trimethylamine and of cesium, two competitive inhibitors. Polarized absorption microspectrophotometry was performed on single crystals under various redox, pH, and salt conditions. The results show that the enzyme is catalytically active in the crystal and that the cations cause the same spectral perturbations as are observed in solution. These studies lead us to propose a model for the entrance and binding of the substrate in the active site.

Proposed steady-state kinetic mechanism for Corynebacterium ammoniagenes FAD synthetase produced by Escherichia coli.

The bifunctional enzyme, FAD synthetase (FS), from Corynebacterium ammoniagenes was overproduced in Escherichia coli and purified, and its steady-state kinetic properties were investigated. Although FMN is an intermediate product in the conversion of riboflavin to FAD, FMN must be released after formation, and then rebind for adenylylation. It was shown that adenylylation of FMN is reversible; FAD and pyrophosphate can be converted to FMN and ATP by the enzyme. In contrast, under the conditions studied, phosphorylation of riboflavin is irreversible. A method is described for analysis of two catalytic cycles, occurring on one enzyme, which have a substrate and/or product in common. The binding order for the phosphorylation cycle of FS was established as riboflavin(in), ATP(in), ADP(out), and FMN(out). The order for the adenylylation cycle was ATP(in), FMN(in), pyrophosphate(out), and FAD(out). A set of steady-state constants was determined, and without additional optimization, these constants were sufficient to describe experimental progress curves for conversion of riboflavin to FAD. In independent studies, it was demonstrated that FMN binds to apo-FS with a dissociation constant of 6-7 microM, which is 2 orders of magnitude higher than the KD value for riboflavin. For the steady-state kinetic analysis, this represents reversible binding of FMN(out) in the phosphorylation cycle (cycle I), which effectively inhibits catalysis in the adenylylation cycle (cycle II).

The cytochrome subunit is necessary for covalent FAD attachment to the flavoprotein subunit of p-cresol methylhydroxylase.

When p-cresol methylhydroxylase (PCMH) is expressed in its natural host Pseudomonas putida, or when the genes of the alpha and beta subunits of the enzyme are expressed together in the heterologous host Escherichia coli, flavin-adenine dinucleotide (FAD) is covalently attached to Tyr384 of the alpha subunit and the correct alpha 2 beta 2 form of the enzyme is assembled. The apoflavoprotein has been expressed in E. coli in the absence of the beta cytochrome c subunit and purified. While noncovalent FAD binding to apoflavoprotein in the absence of the cytochrome subunit could not be directly demonstrated, circumstantial evidence suggests that this indeed occurs. Covalent flavinylation requires one molecule each of FAD and cytochrome for each flavoprotein subunit. The flavinylation process leads to the 2-electron-reduced form of covalently bound FAD, and the resulting alpha 2 beta 2 enzyme is identical to wild-type PCMH. This work presents clear evidence that covalent flavinylation occurs by a self-catalytic mechanism; an external enzyme or chaperon is not required, nor is prior chemical activation of FAD or of the protein. This work is the first to define the basic chemistry of covalent flavinylation of an enzyme to produce the normal, active species, and confirms a long standing, postulated chemical mechanism of this process. It also demonstrates, for the first time, the absolute requirement for a partner subunit in the post-translational modification of a protein. It is proposed that the covalent FAD bond to Tyr384 and the phenolic portion of this Tyr are part of the essential electron transfer path from FAD to heme.

Influence of monovalent cations on the ultraviolet-visible spectrum of tryptophan tryptophylquinone-containing methylamine dehydrogenase from bacterium W3A1.

The influence of the monovalent cations on the UV-visible spectra of the methylamine dehydrogenase (MADH) from bacterium W3A1 was investigated. The spectra for the oxidized and 1- and 2-electron-reduced forms, unperturbed by bound cations, were obtained for the enzyme, and the extinction coefficients for these forms were determined. The binding of the following cations was investigated: Li+, Na+, K+, Rb+, Cs+, NH4+, (CH3)3NH+, and (CH3)4N+. It was shown that each cation produced unique spectral changes, some of which were pH-dependent. Except for NH4+, all spectral changes produced by binding of the monovalent cations can be explained by assuming two different binding sites in MADH (type I and type II sites). Na+ and K+ displayed monophasic binding to the type II site, (CH3)3NH+ and (CH3)4N+ displayed monophasic binding to the type I site, and Li+, Rb+, and Cs+ displayed either monophasic or biphasic binding to one or both sites depending on pH. The pH dependence for binding to the two sites is different, i.e. plots of log(Kd) versus pH have negative slopes approximately 1 for the type II site, whereas the negative slope is significantly less than 1 (0.6-0.8) for the type I site. This difference leads to pH-dependent changes in spectral features produced by binding of Li+, Rb+, and Cs+. The spectral changes seen during titrations with NH+4 were unlike those seen for any other cation. The binding of NH+4 was biphasic, and the spectra produced in each phase were unaffected by pH. It is assumed that this cation binds to the tryptophan tryptophylquinone cofactor to produce the iminoquinone in the first phase and then binds to the type I monovalent cation binding site in the second phase. It is suggested that binding of NH+4 (and CH3NH+3) to the type I site is a prelude to binding to the cofactor.

[Inhibition of cholinesterase activity with fluorine-containing derivatives of alpha-aminophosphonic acid]. / Ingibirovanie aktivnosti kholinésteraz ftorsoderzhashchimi proizvodnymi alpha-aminofosphonovykh kislot.

A series of O,O-diethyl-1-(N-alpha-hydrohexafluoroisobutyryl)aminoalkylphos phonates (APh) has been synthesized and their interaction with human erythrocyte acetylcholinesterase (AChE) and with horse serum butyrylcholinesterase (BuChE) studied. Most of the APhs inactivated the cholinesterases irreversible through formation of the enzyme-inhibitor intermediate. The inactivation rate constants and the enzyme-inhibitor intermediate dissociation constants are calculated. The quantitative structure-activity relationships including both hydrophobic and calculated steric parameters of substituents are developed for APh--ChE interactions. Molecular mechanics (programme MM2) was used for determining steric parameters (Es). On the basis of QSAR models analysis it was concluded that hydrophobic interactions play an essential role in APh--AChE binding, whereas for APh--BuChE binding steric interactions are essential. Presence of at least two APh binding centres on the surface of AChE and BuChE is suggested.