The thiostrepton A tryptophan methyltransferase TsrM catalyses a cob(II)alamin-dependent methyl transfer reaction.
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ABSTRACT:
SUBMITTER: Benjdia A
PROVIDER: S-EPMC4632189 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Methanogenesis from methylated substrates is initiated by substrate-specific methyltransferases that generate the central metabolic intermediate methyl-coenzyme M. This reaction involves a methyl-corrinoid protein intermediate and one or two cognate methyltransferases. Based on genetic data, the Methanosarcina acetivorans MtpC (corrinoid protein) and MtpA (methyltransferase) proteins were suggested to catalyze the methylmercaptopropionate (MMPA):coenzyme M (CoM) methyl transfer reaction without a second methyltransferase. To test this, MtpA was purified after overexpression in its native host and characterized biochemically. MtpA catalyzes a robust methyl transfer reaction using free methylcob(III)alamin as the donor and mercaptopropionate (MPA) as the acceptor, with kcat of 0.315 s-1 and apparent Km for MPA of 12 μM. CoM did not serve as a methyl acceptor; thus, a second unidentified methyltransferase is required to catalyze the full MMPA:CoM methyl transfer reaction. The physiologically relevant methylation of cob(I)alamin with MMPA, which is thermodynamically unfavorable, was also demonstrated, but only at high substrate concentrations. Methylation of cob(I)alamin with methanol, dimethylsulfide, dimethylamine, and methyl-CoM was not observed, even at high substrate concentrations. Although the corrinoid protein MtpC was poorly expressed alone, a stable MtpA/MtpC complex was obtained when both proteins were coexpressed. Biochemical characterization of this complex was not feasible, because the corrinoid cofactor of this complex was in the inactive Co(II) state and was not reactivated by incubation with strong reductants. The MtsF protein, composed of both corrinoid and methyltransferase domains, copurifies with the MtpA/MtpC, suggesting that it may be involved in MMPA metabolism.IMPORTANCE Methylmercaptopropionate (MMPA) is an environmentally significant molecule produced by degradation of the abundant marine metabolite dimethylsulfoniopropionate, which plays a significant role in the biogeochemical cycles of both carbon and sulfur, with ramifications for ecosystem productivity and climate homeostasis. Detailed knowledge of the mechanisms for MMPA production and consumption is key to understanding steady-state levels of this compound in the biosphere. Unfortunately, the biochemistry required for MMPA catabolism under anoxic conditions is poorly characterized. The data reported here validate the suggestion that the MtpA protein catalyzes the first step in the methanogenic catabolism of MMPA. However, the enzyme does not catalyze a proposed second step required to produce the key intermediate, methyl coenzyme M. Therefore, the additional enzymes required for methanogenic MMPA catabolism await discovery.
| S-EPMC6531614 | biostudies-literature
Project description:The one-electron-reduced form of vitamin B(12), cob(II)alamin (Co(2+)Cbl), is found in several essential human enzymes, including the cobalamin-dependent methionine synthase (MetH). In this work, experimentally validated electronic structure descriptions for two "base-off" Co(2+)Cbl species have been generated using a combined spectroscopic and computational approach, so as to obtain definitive clues as to how these and related enzymes catalyze the thermodynamically challenging reduction of Co(2+)Cbl to cob(I)alamin (Co(1+)Cbl). Specifically, electron paramagnetic resonance (EPR), electronic absorption (Abs), and magnetic circular dichroism (MCD) spectroscopic techniques have been employed as complementary tools to characterize the two distinct forms of base-off Co(2+)Cbl that can be trapped in the H759G variant of MetH, one containing a five-coordinate and the other containing a four-coordinate, square-planar Co(2+) center. Accurate spin Hamiltonian parameters for these low-spin Co(2+) centers have been determined by collecting EPR data using both X- and Q-band microwave frequencies, and Abs and MCD spectroscopic techniques have been employed to probe the corrin-centered pi --> pi* and Co-based d --> d excitations, respectively. By using these spectroscopic data to evaluate electronic structure calculations, we found that density functional theory provides a reasonable electronic structure description for the five-coordinate form of base-off Co(2+)Cbl. However, it was necessary to resort to a multireference ab initio treatment to generate a more realistic description of the electronic structure of the four-coordinate form. Consistent with this finding, our computational data indicate that, in the five-coordinate Co(2+)Cbl species, the unpaired spin density is primarily localized in the Co 3d(z(2))-based molecular orbital, as expected, whereas in the four-coordinate form, extensive Co 3d orbital mixing, configuration interaction, and spin-orbit coupling cause the unpaired electron to delocalize over several Co 3d orbitals. These results provide important clues to the mechanism of enzymatic Co(2+)Cbl --> Co(1+)Cbl reduction.
| S-EPMC3127260 | biostudies-literature
Project description:The hydration of the coenzyme cob(II)alamin has been studied using high-resolution monochromatic neutron crystallographic data collected at room temperature to a resolution of 0.92 Å on the original D19 diffractometer with a prototype 4° × 64° detector at the high-flux reactor neutron source run by the Institute Laue-Langevin. The resulting structure provides hydrogen-bonding parameters for the hydration of biomacromolecules to unprecedented accuracy. These experimental parameters will be used to define more accurate force fields for biomacromolecular structure refinement. The presence of a hydrophobic bowl motif surrounded by flexible side chains with terminal functional groups may be significant for the efficient scavenging of ligands. The feasibility of extending the resolution of this structure to ultrahigh resolution was investigated by collecting time-of-flight neutron crystallographic data during commissioning of the TOPAZ diffractometer with a prototype array of 14 modular 2° × 21° detectors at the Spallation Neutron Source run by Oak Ridge National Laboratory.
| S-EPMC3107055 | biostudies-literature
Project description:The electron paramagnetic resonance (EPR) spectrum of an intermediate freeze trapped during the steady state of the reaction catalyzed by the adenosylcobalamin (AdoCbl)-dependent enzyme, methylmalonyl-CoA mutase, has been studied. The EPR spectrum is that of a hybrid triplet spin system created as a result of strong electron-electron spin coupling between an organic radical and the low-spin Co(2+) in cob(II)alamin. The spectrum was analyzed by simulation to obtain the zero-field splitting (ZFS) parameters and Euler angles relating the radical-to-cobalt interspin vector to the g axis system of the low-spin Co(2+). Labeling of the substrate with (13)C and (2)H was used to probe the identity of the organic radical partner in the triplet spin system. The patterns of inhomogeneous broadening in the EPR signals produced by [2'-(13)C]methylmalonyl-CoA and [2-(13)C]methylmalonyl-CoA as well as line narrowing resulting from deuterium substitution in the substrate were consistent with those expected for a succinyl-CoA radical wherein the unpaired electron was centered on the carbon alpha to the free carboxyate group of the rearranged radical. The interspin distance and the Euler angles were used to position this product radical into the active site of the enzyme.
| S-EPMC3132558 | biostudies-literature
Project description:O?.- scavenger: The rate constant for the rapid reaction of the ROS superoxide with the reduced vitamin B?? radical complex cob(II)alamin was directly determined to be 3.8×10(8) M?¹ s?¹. This rate was independent of pH over the range 5.5-8.7. These results have implications for studying the use of B?? supplements to combat diseases associated with oxidative stress.
| S-EPMC3737425 | biostudies-other
Project description:The methyltransferase FliB posttranslationally modifies surface-exposed ɛ-N-lysine residues of flagellin, the protomer of the flagellar filament in Salmonella enterica (S. enterica). Flagellin methylation, reported originally in 1959, was recently shown to enhance host cell adhesion and invasion by increasing the flagellar hydrophobicity. The role of FliB in this process, however, remained enigmatic. In this study, we investigated the properties and mechanisms of FliB from S. enterica in vivo and in vitro. We show that FliB is an S-adenosylmethionine (SAM) dependent methyltransferase, forming a membrane associated oligomer that modifies flagellin in the bacterial cytosol. Using X-band electron paramagnetic resonance (EPR) spectroscopy, zero-field 57Fe Mössbauer spectroscopy, methylation assays and chromatography coupled mass spectrometry (MS) analysis, we further found that FliB contains an oxygen sensitive [4Fe-4S] cluster that is essential for the methyl transfer reaction and might mediate a radical mechanism. Our data indicate that the [4Fe-4S] cluster is coordinated by a cysteine rich motif in FliB that is highly conserved among multiple genera of the Enterobacteriaceae family.
| S-EPMC8598068 | biostudies-literature
Project description:Solvent (e.g., water)-catalyzed proton transfer (SCPT) via the relay of hydrogen (H)-bonds plays a key role in proton migration. In this study, a new class of 1H-pyrrolo[3,2-g]quinolines (PyrQs) and their derivatives were synthesized, with sufficient separation of the pyrrolic proton donating and pyridinic proton accepting sites to probe excited-state SCPT. There was dual fluorescence for all PyrQs in methanol, i.e., normal (PyrQ) and tautomer 8H-pyrrolo[3,2-g]quinoline (8H-PyrQ) emissions. The fluorescence dynamics unveiled a precursor (PyrQ) and successor (8H-PyrQ) relationship and the correlation of an increasing overall excited-state SCPT rate (kSCPT) upon increasing the N(8)-site basicity. kSCPT can be expressed by the coupling reaction kSCPT = Keq × kPT, where kPT denotes the intrinsic proton tunneling rate in the relay and Keq denotes the pre-equilibrium between randomly and cyclically H-bonded solvated PyrQs. Molecular dynamics (MD) simulation defined the cyclic PyrQs and analyzed the H-bond and molecular arrangement over time, which showed the cyclic PyrQs incorporating ≧3 methanol molecules. These cyclic H-bonded PyrQs are endowed with a relay-like proton transfer rate, kPT. MD simulation estimated an upper-limited Keq value of 0.02-0.03 for all studied PyrQs. When there was little change in Keq, the distinct kSCPT values for PyrQs were at different kPT values, which increased as the N(8) basicity increased, which was induced by the C(3)-substituent. kSCPT was subject to a deuterium isotope effect, where the kSCPT of 1.35 × 1010 s-1 for PyrQ-D in CH3OD was 1.68 times slower than that (2.27 × 1010 s-1) of PyrQ in CH3OH. MD simulation provided a similar Keq for PyrQ and PyrQ-D, leading to different proton tunneling rates (kPT) between PyrQ and PyrQ-D.
| S-EPMC10321479 | biostudies-literature
Project description:CobA from Salmonella enterica (SeCobA) is a member of the family of ATP:Co(I)rrinoid adenosyltransferase (ACAT) enzymes that participate in the biosynthesis of adenosylcobalamin by catalyzing the transfer of the adenosyl group from an ATP molecule to a reactive Co(I)rrinoid species transiently generated in the enzyme active site. This reaction is thermodynamically challenging, as the reduction potential of the Co(II)rrinoid precursor in solution is far more negative than that of available reducing agents in the cell (e.g., flavodoxin), precluding nonenzymic reduction to the Co(I) oxidation state. However, in the active sites of ACATs, the Co(II)/Co(I) redox potential is increased by >250 mV via the formation of a unique four-coordinate (4c) Co(II)rrinoid species. In the case of the SeCobA ACAT, crystallographic and kinetic studies have revealed that the phenylalanine 91 (F91) and tryptophan 93 (W93) residues are critical for in vivo activity, presumably by blocking access to the lower axial ligand site of the Co(II)rrinoid substrate. To further assess the importance of the F91 and W93 residues with respect to enzymatic function, we have characterized various SeCobA active-site variants using electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopies. Our data provide unprecedented insight into the mechanism by which SeCobA converts the Co(II)rrinoid substrate to 4c species, with the hydrophobicity, size, and ability to participate in offset π-stacking interactions of key active-site residues all being critical for activity. The structural changes that occur upon Co(II)rrinoid binding also appear to be crucial for properly orienting the transiently generated Co(I) "supernucleophile" for rapid reaction with cosubstrate ATP.
| S-EPMC4278676 | biostudies-literature
Project description:The EutT enzyme from Listeria monocytogenes ( LmEutT) is a member of the family of ATP:cobalt(I) corrinoid adenosyltransferase (ACAT) enzymes that catalyze the biosynthesis of adenosylcobalamin (AdoCbl) from exogenous Co(II)rrinoids and ATP. Apart from EutT-type ACATs, two evolutionary unrelated types of ACATs have been identified, termed PduO and CobA. Although the three types of ACATs are nonhomologous, they all generate a four-coordinate cob(II)alamin (4C Co(II)Cbl) species to facilitate the formation of a supernucleophilic Co(I)Cbl intermediate capable of attacking the 5'-carbon of cosubstrate ATP. Previous spectroscopic studies of the EutT ACAT from Salmonella enterica ( SeEutT) revealed that this enzyme requires a divalent metal cofactor for the conversion of 5C Co(II)Cbl to a 4C species. Interestingly, LmEutT does not require a divalent metal cofactor for catalytic activity, which exemplifies an interesting phylogenetic divergence among the EutT enzymes. To explore if this disparity in the metal cofactor requirement among EutT enzymes correlates with differences in substrate specificity or the mechanism of Co(II)Cbl reduction, we employed various spectroscopic techniques to probe the interaction of Co(II)Cbl and cob(II)inamide (Co(II)Cbi+) with LmEutT in the absence and presence of cosubstrate ATP. Our data indicate that LmEutT displays a similar substrate specificity as SeEutT and can bind both Co(II)Cbl and Co(II)Cbi+ when complexed with MgATP, though it exclusively converts Co(II)Cbl to a 4C species. Notably, LmEutT is the most effective ACAT studied to date in generating the catalytically relevant 4C Co(II)Cbl species, achieving a >98% 5C → 4C conversion yield on the addition of just over one mol equiv of cosubstrate MgATP.
| S-EPMC6118401 | biostudies-literature
Project description:Succinyl(carbadethia)-coenzyme A, a synthetic substrate for adenosylcobalamin-dependent methylmalonyl-CoA mutase, has been prepared by a simplified procedure. When recombinant mutase was mixed with the synthetic substrate, the u.v./visible absorption spectrum of the bound cofactor changed rapidly to resemble that of cob(II)alamin, with an absorption maximum at 467 nm. Addition of the natural substrates, in contrast, produced only minor changes in the u.v./visible spectrum. The recent report of the generation of a complex e.p.r. spectrum on addition of substrate to the holo-methylmalonyl-CoA mutase was confirmed with the recombinant enzyme. The signals observed were stronger when the succinyl(carbadethia) analogue was used. Cobalt K-edge X-ray absorption spectroscopy confirmed that the addition of this analogue to holoenzyme leads to the generation of a cob(II)alamin-like species. These results strongly support the generation of cob(II)alamin during the 1,2-skeletal rearrangement catalysed by methylmalonyl-CoA mutase, as required if this enzyme follows the reaction pathway involving radical intermediates previously proposed for other adenosylcobalamin-dependent enzymes.
| S-EPMC1134893 | biostudies-other