pH and deuterium isotope effects on the reaction of trimethylamine dehydrogenase with dimethylamine.

The flavoprotein trimethylamine dehydrogenase is a member of a small class of flavoproteins that catalyze amine oxidation and transfer the electrons through an Fe/S center to an external oxidant. The mechanism of amine oxidation by this family of enzymes has not been established. Here, we describe the use of pH and kinetic isotope effects with the slow substrate dimethylamine to study the mechanism. The data are consistent with the neutral amine being the form of the substrate that binds productively at the pH optimum, since the pKa seen in the kcat/Kamine pH profile for a group that must be unprotonated matches the pKa of dimethylamine. The D(kcat/Kamine) value decreases to unity as the pH decreases. This suggests the presence of an alternative pathway at low pH, in which the protonated substrate binds and is then deprotonated by an active-site residue prior to oxidation. The kcat and Dkcat values both decrease to limiting values at low pH with similar pKa values. This is consistent with a step other than amine oxidation becoming rate-limiting for turnover.

Highly selective ligand binding by Methylophilus methylotrophus cytochrome c''.

Cytochrome c'' (cyt c'') from Methylophilus methylotrophus is unusual insofar as the heme has two axial histidine ligands in the oxidized form but one is detached when the protein is reduced. Despite cyt c'' having an axial site available for binding small ligands, we show here that only NO binds readily to the ferrous cyt c''. Binding of CO, as well as CN(-), on the other hand requires considerable structural reorganization, or reduction of the disulfide bridge close to the heme. Standard free energies for the binding of NO and CO reveal high selectivity of the ferrous cyt c'' for NO, indicating its putative physiological role. In this work, we characterize in detail the kinetics of NO binding and the structural features of the Fe(2+)-NO adduct by stopped-flow and resonance Raman spectroscopy, respectively.

Functional analysis of MmeI from methanol utilizer Methylophilus methylotrophus, a subtype IIC restriction-modification enzyme related to type I enzymes.

MmeI from Methylophilus methylotrophus belongs to the type II restriction-modification enzymes. It recognizes an asymmetric DNA sequence, 5'-TCCRAC-3' (R indicates G or A), and cuts both strands at fixed positions downstream of the specific site. This particular feature has been exploited in transcript profiling of complex genomes (using serial analysis of gene expression technology). We have shown previously that the endonucleolytic activity of MmeI is strongly dependent on the presence of S-adenosyl-l-methionine (J. Nakonieczna, J. W. Zmijewski, B. Banecki, and A. J. Podhajska, Mol. Biotechnol. 37:127-135, 2007), which puts MmeI in subtype IIG. The same cofactor is used by MmeI as a methyl group donor for modification of an adenine in the upper strand of the recognition site to N(6)-methyladenine. Both enzymatic activities reside in a single polypeptide (919 amino acids [aa]), which puts MmeI also in subtype IIC of the restriction-modification systems. Based on a molecular model, generated with the use of bioinformatic tools and validated by site-directed mutagenesis, we were able to localize three functional domains in the structure of the MmeI enzyme: (i) the N-terminal portion containing the endonucleolytic domain with the catalytic Mg2+-binding motif D(70)-X(9)-EXK(82), characteristic for the PD-(D/E)XK superfamily of nucleases; (ii) a central portion (aa 310 to 610) containing nine sequence motifs conserved among N(6)-adenine gamma-class DNA methyltransferases; (iii) the C-terminal portion (aa 610 to 919) containing a putative target recognition domain. Interestingly, all three domains showed highest similarity to the corresponding elements of type I enzymes rather than to classical type II enzymes. We have found that MmeI variants deficient in restriction activity (D70A, E80A, and K82A) can bind and methylate specific nucleotide sequence. This suggests that domains of MmeI responsible for DNA restriction and modification can act independently. Moreover, we have shown that a single amino acid residue substitution within the putative target recognition domain (S807A) resulted in a MmeI variant with a higher endonucleolytic activity than the wild-type enzyme.

Disruption of metF increased L-lysine production by Methylophilus methylotrophus from methanol.

Methionine auxotrophic mutants of Methylophilus methylotrophus AS1 expressing a mutant form of dapA (dapA24) encoding a dihydrodipicolinate synthase desensitized from feedback inhibition by L-lysine, and mutated lysE (lysE24) encoding the L-lysine exporter from Corynebacterium glutamicum 2256, produced higher amounts of L-lysine from methanol as sole carbon source than did other amino acid auxotrophic mutants. Especially, the M. methylotrophus 102 strain, carrying both dapA24 and lysE24, produced L-lysine in more than 1.5 times amounts higher than the parent. A single-base substitution was identified in this auxotroph in codon-329 of the open reading frame of metF, encoding 5,10-methylene-tetra-hydrofolate reductase. We constructed a metF disruptant mutant carrying both dapA24 and lysE24, and confirmed increases in L-lysine production. This is the first report to the effect that metF deficient increased L-lysine production in methylotroph.

L-Lysine biosynthetic pathway of Methylophilus methylotrophus and construction of an L-lysine producer.

Previously, we showed that the enzymes aspartokinase (AK) and dihydrodipicolinate synthase (DDPS), which are involved in L-lysine biosynthesis in the Gram-negative obligate methylotroph Methylophilus methylotrophus AS1, were inhibited by allosteric effectors, including L-lysine. To elucidate further the regulation of L-lysine biosynthesis in M. methylotrophus, we cloned the genes encoding three other enzymes involved in this pathway, L-aspartate-beta-semialdehyde dehydrogenase, dihydrodipicolinate reductase (DDPR) and diaminopimelate decarboxylase, and examined their properties. DDPR was markedly inhibited by L-lysine. Based on this and our previous results, we constructed an L-lysine-producing strain of M. methylotrophus by introducing well-characterized genes encoding desensitized forms of AK and DDPS, as well as dapB (encoding DDPR) from Escherichia coli, using a broad host range plasmid. L-Lysine production was significantly increased by employing an S-(2-aminoethyl)-L-cysteine (L-lysine analog)-resistant mutant as the host. This derivative accumulated L-lysine at a concentration of 1 g l(-1) of medium using methanol as a carbon source.

Kinetic isotope effects and ligand binding in PQQ-dependent methanol dehydrogenase.

The reaction of PQQ (2,7,9-tricarboxypyrroloquinoline quinone)-dependent MDH (methanol dehydrogenase) from Methylophilus methylotrophus has been studied under steady-state conditions in the presence of an alternative activator [GEE (glycine ethyl ester)] and compared with similar reactions performed with ammonium (used more generally as an activator in steady-state analysis of MDH). Studies of initial velocity with methanol (protiated methanol, C1H3O1H) and [2H]methanol (deuteriated methanol, C2H3O2H) as substrate, performed with different concentrations of GEE and PES (phenazine ethosulphate), indicate competitive binding effects for substrate and PES on the stimulation and inhibition of enzyme activity by GEE. GEE is more effective at stimulating activity than ammonium at low concentrations, suggesting tighter binding of GEE to the active site. Inhibition of activity at high GEE concentration is less pronounced than at high ammonium concentration. This suggests a close spatial relationship between the stimulatory (KS) and inhibitory (KI) binding sites in that binding of GEE to the KS site sterically impairs the binding of GEE to the KI site. The binding of GEE is also competitive with the binding of PES, and GEE is more effective than ammonium in competing with PES. This competitive binding of GEE and PES lowers the effective concentration of PES at the site competent for electron transfer. Accordingly, the oxidative half-reaction, which is second-order with respect to PES concentration, is more rate-limiting in steady-state turnover with GEE than with ammonium. The smaller methanol C-1H/C-2H kinetic isotope effects observed with GEE are consistent with a larger contribution made by the oxidative half-reaction to rate limitation. Cyanide is much less effective at suppressing 'endogenous' activity in the presence of GEE than with ammonium, which is attributed to impaired binding of cyanide to the catalytic site through steric interaction with GEE bound at the KS site. The kinetic model developed previously for reactions of MDH with ammonium [Hothi, Basran, Sutcliffe and Scrutton (2003) Biochemistry 42, 3966-3978] is consistent with data obtained with GEE, although a more detailed structural interpretation is given here. Molecular-modelling studies rationalize the kinetic observations in terms of a complex binding scenario at the molecular level involving two spatially distinct inhibitory sites (KI and KI'). The KI' site caps the entrance to the active site and is interpreted as the PES binding site. The KI site is adjacent to, and, for GEE, overlaps with, the KS site, and is located in the active-site cavity close to the PQQ cofactor and the catalytic site for methanol oxidation.

[The release of flavin adenine dinucleotide upon local conformational transition in electron-transferring flavoprotein induced by trimethylamine dehydrogenase]. / Vysvobozhdenie FAD pri lokal'nom konformatsionnom perekhode, indutsiruemom trimetilaminodegidrogenazoi v elektrontransportnom flavoproteine.

The electron-transferring proteins, trimethylamine dehydrogenase (TMAD) and electron-transferring flavoprotein (ETF) from the bacterium Methylophilius methylotrophus, were studied in vitro by fluorescence spectroscopy. Flavin adenine dinucleotide (FAD) was found to be capable of a slow and spontaneous release from ETF, which is accompanied by an increase in flavin fluorescence. At a rather high ionic strength (0.1 M NaCl or 50 mM phosphate), the FAD release is sharply activated by TMAD preparations that induce a local conformational transition in ETF. The values of tryptophan fluorescence polarization and lifetime and the use of the Levshin-Perrin equation helped show that the size of protein particles remain unchanged upon the TMAD and ETF mixing; i.e., these proteins themselves do not form a stable complex with each other. The protein mixture did not release flavin from ETF in the presence of trimethylamine and formaldehyde. In this case, a stable complex between the proteins appeared to be formed under the action of formaldehyde. Upon a short-term incubation of ETF with ferricyanide, FAD was hydrolyzed to flavin mononucleotide (FMN) and AMP. This fact explains the previous detection of AMP in ETF preparations by some researches. A fluorescence method was proposed for distinguishing FAD from FMN in solution using ethylene glycol. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.

Crystallization and preliminary X-ray characterization of cytochrome c" from the obligate methylotroph Methylophilus methylotrophus.

Cytochrome c" from the obligate methylotroph Methylophilus methylotrophus is a 15 kDa monohaem protein which has a c-type haem covalently linked to the protein chain. Two histidine residues are the axial ligands of the Fe atom in the oxidized form. This cytochrome is one of the few known haem proteins which undergoes a change of spin state of the Fe atom upon reduction, with the detachment of an axial histidine ligand. Initial crystallization conditions involved the utilization of cadmium chloride as an additive and resulted in highly mosaic crystals with poor diffraction properties. Optimization of the crystallization conditions was achieved by slowing the nucleation process utilizing agarose gels and viscous additives such as PEG, ethylene glycol and glycerol. Addition of glycerol to the crystallization buffer produced crystals suitable for X-ray diffraction, with a reduced solvent content and mosaicity, which diffracted to a maximum resolution of 1.19 A using synchrotron radiation. The crystals obtained under these conditions were employed for structure solution using the multiwavelength anomalous dispersion method at the Fe K edge.

Effect of pH on axial ligand coordination of cytochrome c" from Methylophilus methylotrophus and horse heart cytochrome c.

The effect of protons on the axial ligand coordination and on structural aspects of the protein moiety of cytochrome c' ' from Methylophilus methylotrophus, an obligate methylotroph, has been investigated down to very low pH (i.e., 0.3). The unusual resistance of this cytochrome to very low pH values has been exploited to carry out this study in comparison with horse heart cytochrome c. The experiments were undertaken at a constant phosphate concentration to minimize the variation of ionic strength with pH. The pH-linked effects have been monitored at 23 degrees C in the oxidized forms of both cytochromes by following the variations in the electronic absorption, circular dichroism and resonance Raman spectra. This approach has enabled the conformational changes of the heme surroundings to be monitored and compared with the concomitant overall structural rearrangements of the molecule. The results indicate that horse heart cytochrome c undergoes a first conformational change at around pH 2.0. This event is possibly related to the cleavage of the Fe-Met80 bond and a likely coordination of a H(2)O molecule as a sixth axial ligand. Conversely, in cytochrome c" from M. methylotrophus, a variation of the axial ligand coordination occurs at a pH that is about 1 unit lower. Further, it appears that a concerted cleavage of both His ligands takes place, suggesting indeed that the different axial ligands present in horse heart cytochrome c (Met/His) and in cytochrome c" from M. methylotrophus (His/His) affect the heme conformational changes.

Radiolytic studies of trimethylamine dehydrogenase. Spectral deconvolution of the neutral and anionic flavin semiquinone, and determination of rate constants for electron transfer in the one-electron reduced enzyme.

Trimethylamine dehydrogenase from the pseudomonad Methylophilus methylotrophus has been examined using the technique of pulse radiolysis to rapidly introduce a single reducing equivalent into the enzyme. Using enzyme that has had its iron-sulfur center rendered redox-inert by prior reaction with ferricenium hexafluorophosphate, we determined the spectral change associated with formation of both the anionic and neutral forms that were generated at high and low pH, respectively, of the unique 6-cysteinyl-FMN of the enzyme. With native enzyme, electron transfer was observed within the radiolytically generated one-electron reduced enzyme but only at low pH (6.0). The kinetics and thermodynamics of this electron transfer in one-electron reduced enzyme may be compared with that studied previously in the two-electron reduced enzyme. In contrast to previous studies with two-electron reduced enzyme in which a pK(a) of approximately 8 was determined for the flavin semiquinone, in the one-electron reduced enzyme the semiquinone was not substantially protonated even at pH 6. 0. These results indicate that reduction of the iron-sulfur center of the enzyme significantly decreases the pK(a) of the flavin semiquinone of the active site. This provides further evidence, in conjunction with the strong magnetic interaction known to exist between the centers in the two-electron reduced enzyme, that the two redox-active centers in trimethylamine dehydrogenase are in intimate contact with one another in the active site of the enzyme.

Differential coupling through Val-344 and Tyr-442 of trimethylamine dehydrogenase in electron transfer reactions with ferricenium ions and electron transferring flavoprotein.

Modeling studies of the trimethylamine dehydrogenase-electron transferring flavoprotein (TMADH-ETF) electron transfer complex have suggested potential roles for Val-344 and Tyr-442, found on the surface of TMADH, in electronic coupling between the 4Fe-4S center of TMADH and the FAD of ETF. The importance of these residues in electron transfer, both to ETF and to the artificial electron acceptor, ferricenium (Fc(+)), has been studied by site-directed mutagenesis and stopped-flow spectroscopy. Reduction of the 6-(S)-cysteinyl FMN in TMADH is not affected by mutation of either Tyr-442 or Val-344 to a variety of alternate side chains, although there are modest changes in the rate of internal electron transfer from the 6-(S)-cysteinyl FMN to the 4Fe-4S center. The kinetics of electron transfer from the 4Fe-4S center to Fc(+) are sensitive to mutations at position 344. The introduction of smaller side chains (Ala-344, Cys-344, and Gly-344) leads to enhanced rates of electron transfer, and likely reflects shortened electron transfer "pathways" from the 4Fe-4S center to Fc(+). The introduction of larger side chains (Ile-344 and Tyr-344) reduces substantially the rate of electron transfer to Fc(+). Electron transfer to ETF is not affected, to any large extent, by mutation of Val-344. In contrast, mutation of Tyr-442 to Phe, Leu, Cys, and Gly leads to major reductions in the rate of electron transfer to ETF, but not to Fc(+). The data indicate that electron transfer to Fc(+) is via the shortest pathway from the 4Fe-4S center of TMADH to the surface of the enzyme. Val-344 is located at the end of this pathway at the bottom of a small groove on the surface of TMADH, and Fc(+) can penetrate this groove to facilitate good electronic coupling with the 4Fe-4S center. With ETF as an electron acceptor, the observed rate of electron transfer is substantially reduced on mutation of Tyr-442, but not Val-344. We conclude that the flavin of ETF does not penetrate fully the groove on the surface of TMADH, and that electron transfer from the 4Fe-4S center to ETF may involve a longer pathway involving Tyr-442. Mutation of Tyr-442 likely disrupts electron transfer by perturbing the interaction geometry of TMADH and ETF in the productive electron transfer complex, leading to less efficient coupling between the redox centers.

Structural and biochemical characterization of recombinant wild type and a C30A mutant of trimethylamine dehydrogenase from methylophilus methylotrophus (sp. W(3)A(1)).

Trimethylamine dehydrogenase (TMADH) is an iron-sulfur flavoprotein that catalyzes the oxidative demethylation of trimethylamine to form dimethylamine and formaldehyde. It contains a unique flavin, in the form of a 6-S-cysteinyl FMN, which is bent by approximately 25 degrees along the N5-N10 axis of the flavin isoalloxazine ring. This unusual conformation is thought to modulate the properties of the flavin to facilitate catalysis, and has been postulated to be the result of covalent linkage to Cys-30 at the flavin C6 atom. We report here the crystal structures of recombinant wild-type and the C30A mutant TMADH enzymes, both determined at 2.2 A resolution. Combined crystallographic and NMR studies reveal the presence of inorganic phosphate in the FMN binding site in the deflavo fraction of both recombinant wild-type and C30A proteins. The presence of tightly bound inorganic phosphate in the recombinant enzymes explains the inability to reconstitute the deflavo forms of the recombinant wild-type and C30A enzymes that are generated in vivo. The active site structure and flavin conformation in C30A TMADH are identical to those in recombinant and native TMADH, thus revealing that, contrary to expectation, the 6-S-cysteinyl FMN link is not responsible for the 25 degrees butterfly bending along the N5-N10 axis of the flavin in TMADH. Computational quantum chemistry studies strongly support the proposed role of the butterfly bend in modulating the redox properties of the flavin. Solution studies reveal major differences in the kinetic behavior of the wild-type and C30A proteins. Computational studies reveal a hitherto, unrecognized, contribution made by the S(gamma) atom of Cys-30 to substrate binding, and a role for Cys-30 in the optimal geometrical alignment of substrate with the 6-S-cysteinyl FMN in the enzyme active site.

A mechanism for substrate-Induced formation of 6-hydroxyflavin mononucleotide catalyzed by C30A trimethylamine dehydrogenase.

Experiments are described to determine the origin of the 6-hydroxyl group of 6-hydroxyFMN produced by the substrate-induced transformation of FMN in the C30A mutant of trimethylamine dehydrogenase. The conversion of FMN to 6-hydroxyFMN is carried out in the presence of H(2)(18)O and 18O(2), and the results clearly show that the 6-hydroxyl group is derived from molecular oxygen and not from water.

Effects of multiple ligand binding on kinetic isotope effects in PQQ-dependent methanol dehydrogenase.

The reaction of PQQ-dependent methanol dehydrogenase (MDH) from Methylophilus methylotrophus has been studied by steady-state and stopped-flow kinetic methods, with particular reference to multiple ligand binding and the kinetic isotope effect (KIE) for PQQ reduction. Phenazine ethosulfate (PES; an artificial electron acceptor) and cyanide (a suppressant of endogenous activity), but not ammonium (an activator of MDH), compete for binding at the catalytic methanol-binding site. Cyanide does not activate turnover in M. methylotrophus MDH, as reported previously for the Paracoccus denitrificans enzyme. Activity is dependent on activation by ammonium but is inhibited at high ammonium concentrations. PES and methanol also influence the stimulatory and inhibitory effects of ammonium through competitive binding. Reaction profiles as a function of ammonium and PES concentration differ between methanol and deuterated methanol, owing to force constant effects on the binding of methanol to the stimulatory and inhibitory ammonium binding sites. Differential binding gives rise to unusual KIEs for PQQ reduction as a function of ammonium and PES concentration. The observed KIEs at different ligand concentrations are independent of temperature, consistent with their origin in differential binding affinities of protiated and deuterated substrate at the ammonium binding sites. Stopped-flow studies indicate that enzyme oxidation is not rate-limiting at low ammonium concentrations (<4 mM) during steady-state turnover. At higher ammonium concentrations (>20 mM), the low effective concentration of PES in the active site owing to the competitive binding of ammonium lowers the second-order rate constant for enzyme oxidation, and the oxidative half-reaction becomes more rate limiting. A sequential stopped-flow method is reported that has enabled, for the first time, a detailed study of the reductive half-reaction of MDH and comparison with steady-state data. The limiting rate of PQQ reduction (0.48 s(-1)) is less than the steady-state turnover number, and the observed KIE in stopped-flow studies is unity. Although catalytically active, we propose reduction of the oxidized enzyme generated in stopped-flow analyses is gated by conformational change or ligand exchange. Slow recovery from this trapped state on mixing with methanol accounts for the slow reduction of PQQ and a KIE of 1. This study emphasizes the need for caution in using inflated KIEs, and the temperature dependence of KIEs, as a probe for hydrogen tunneling.

Characterization of the L-lysine biosynthetic pathway in the obligate methylotroph Methylophilus methylotrophus.

The L-lysine biosynthetic pathway of the gram-negative obligate methylotroph Methylophilus methylotrophus AS1 was examined through characterization of the enzymes aspartokinase (AK), aspartsemialdehyde dehydrogenase, dihydrodipicolinate synthase (DDPS), dihydrodipicolinate reductase, and diaminopimelate decarboxylase. The AK was inhibited by L-threonine and by a combination of L-threonine and L-lysine, but not by L-lysine alone, and the activity of DDPS was moderately reduced by L-lysine. In an L-lysine producing mutant (G49), isolated as an S-(2-aminoethyl)-L-cysteine (lysine analog) resistant strain, both AK and DDPS were partially resistant to feedback inhibition. The ask and dapA genes encoding AK and DDPS respectively were isolated from the parental strain, AS1, and its G49 derivative. Comparison of the sequences revealed a point mutation in each of these genes in G49. The mutation in the ask gene altered aspartic acid in a key region involved in the allosteric regulation common to AKs, while a novel mutation in the dapA gene altered tyrosine-106, which was assumed to be involved in the binding of L-lysine to DDPS.