Isotope labeling studies reveal the order of oxygen incorporation into the tryptophan tryptophylquinone cofactor of methylamine dehydrogenase

The bacterial enzyme methylamine dehydrogenase (MADH) contains the protein-derived cofactor tryptophan tryptophylquinone (TTQ). TTQ is generated by the posttranslational modification of two endogenous MADH tryptophans, betaW57 and betaW108. Two oxygens are inserted sequentially into betaW57 to generate the quinone moiety, after which it is cross-linked to betaW108. We have previously shown that the second oxygenation and cross-link formation are catalyzed by the novel di-heme cytochrome MauG. Here we show, using isotopically labeled oxygen and water, that the first addition of oxygen occurs specifically at the C7 position of betaW57 and that MauG then inserts the second oxygen at position C6.     

Analysis of classical and quantum paths for deprotonation of methylamine by methylamine dehydrogenase.

The hydrogen-transfer reaction catalysed by methylamine dehydrogenase (MADH) with methylamine (MA) as substrate is a good model system for studies of proton tunnelling in enzyme reactions--an area of great current interest--for which atomistic simulations will be vital. Here, we present a detailed analysis of the key deprotonation step of the MADH/MA reaction and compare the results with experimental observations. Moreover, we compare this reaction with the related aromatic amine dehydrogenase (AADH) reaction with tryptamine, recently studied by us, and identify possible causes for the differences observed in the measured kinetic isotope effects (KIEs) of the two systems. We have used combined quantum mechanics/molecular mechanics (QM/MM) techniques in molecular dynamics simulations and variational transition state theory with multidimensional tunnelling calculations averaged over an ensemble of paths. The results reveal important mechanistic complexity. We calculate activation barriers and KIEs for the two possible proton transfers identified-to either of the carboxylate oxygen atoms of the catalytic base (Asp428beta)-and analyse the contributions of quantum effects. The activation barriers and tunnelling contributions for the two possible proton transfers are similar and lead to a phenomenological activation free energy of 16.5+/-0.9 kcal mol(-1) for transfer to either oxygen (PM3-CHARMM calculations applying PM3-SRP specific reaction parameters), in good agreement with the experimental value of 14.4 kcal mol(-1). In contrast, for the AADH system, transfer to the equivalent OD1 was found to be preferred. The structures of the enzyme complexes during reaction are analysed in detail. The hydrogen bond of Thr474beta(MADH)/Thr172beta(AADH) to the catalytic carboxylate group and the nonconserved active site residue Tyr471beta(MADH)/Phe169beta(AADH) are identified as important factors in determining the preferred oxygen acceptor. The protein environment has a significant effect on the reaction energetics and hence on tunnelling contributions and KIEs. These environmental effects, and the related clearly different preferences for the two carboxylate oxygen atoms (with different KIEs) in MADH/MA and AADH/tryptamine, are possible causes of the differences observed in the KIEs between these two important enzyme reactions.     

Evidence for substrate activation of electron transfer from methylamine dehydrogenase to amicyanin

Electron transfer (ET) from methylamine dehydrogenase (MADH) to amicyanin may be true or gated ET, depending upon the redox form of MADH. ET from the substrate-reduced aminoquinol form of MADH is gated, and the reaction rate is dependent on the presence of monovalent cations. This ET reaction has been studied in buffer free of monovalent cations. The reaction rate is orders of magnitude less than with saturating concentrations of monovalent cation. Analysis of the temperature dependence of this slow reaction, however, reveals that it is a true ET reaction. The rate of MADH reduction by substrate and the steady-state rate of substrate-dependent reduction of amicyanin by MADH were examined in different buffers. The results reveal that, in the steady state, the protonated methylammonium substrate performs the role previously attributed to monovalent cations in regulating the rate and mechanism of ET from MADH. The two putative cation binding sites previously observed in the crystal structure of MADH may now be assigned distinct roles, one as a catalytic substrate binding site and the other as a noncatalytic regulatory substrate binding site.     

Molecular dynamics study of the active site of methylamine dehydrogenase

We have obtained AMBER94 force-field parameters for the TTQ cofactor of the enzyme methylamine dehydrogenase (MADH). This enzyme catalyzes the oxidation of methylamine to produce formaldehyde and ammonia. In the rate-determining step of the catalyzed reaction, a proton is transferred from the methyl group of the substrate to residue Asp76. We used the new parameters to perform molecular dynamics simulations of MADH in order to characterize the dynamics of the active site prior to the proton-transfer step. We found that only one of the oxygen atoms of Asp76 can act as an acceptor of the proton. The other oxygen interacts with Thr122 via a strong hydrogen bond. In contrast, because of the rotation the methyl group of the substrate, the three methyl hydrogen atoms are alternately in position to be transferred. The distance that the proton has to travel presents a broad distribution with a peak between 1.0 and 1.1 A and reaches values as short as 0.8 A. The fluctuation of the distance between the donor and the acceptor has the largest frequency component at 50 cm(-1), but the spectrum presents a rich structure between 10 and 400 cm(-1). The more important peaks appear below 250 cm(-1).     

Probing Bis‐FeIV MauG: Experimental Evidence for the Long‐Range Charge‐Resonance Model

The biosynthesis of tryptophan tryptophylquinone, a protein‐derived cofactor, involves a long‐range reaction mediated by a bis‐Fe^IV intermediate of a diheme enzyme, MauG. Recently, a unique charge‐resonance (CR) phenomenon was discovered in this intermediate, and a biological, long‐distance CR model was proposed. This model suggests that the chemical nature of the bis‐Fe^IV species is not as simple as it appears; rather, it is composed of a collection of resonance structures in a dynamic equilibrium. Here, we experimentally evaluated the proposed CR model by introducing small molecules to, and measuring the temperature dependence of, bis‐Fe^IV MauG. Spectroscopic evidence was presented to demonstrate that the selected compounds increase the decay rate of the bis‐Fe^IV species by disrupting the equilibrium of the resonance structures that constitutes the proposed CR model. The results support this new CR model and bring a fresh concept to the classical CR theory.     

Sequential enzymatic epoxidation involved in polyether lasalocid biosynthesis

Enantioselective epoxidation followed by regioselective epoxide opening reaction are the key processes in construction of the polyether skeleton. Recent genetic analysis of ionophore polyether biosynthetic gene clusters suggested that flavin-containing monooxygenases (FMOs) could be involved in the oxidation steps. In vivo and in vitro analyses of Lsd18, an FMO involved in the biosynthesis of polyether lasalocid, using simple olefin or truncated diene of a putative substrate as substrate mimics demonstrated that enantioselective epoxidation affords natural type mono- or bis-epoxide in a stepwise manner. These findings allow us to figure out enzymatic polyether construction in lasalocid biosynthesis.     

CmlI is an N-oxygenase in the biosynthesis of chloramphenicol

The N-oxygenation of an amine group is one of the steps in the biosynthesis of the antibiotic chloramphenicol. The non-heme di-iron enzyme CmlI was identified as the enzyme catalyzing this reaction through bioinformatics studies and reconstitution of enzymatic activity. In vitro reconstitution was achieved using phenazine methosulfate and NADH as electron mediators, while in vivo activity was demonstrated in Escherichia coli using two substrates. Kinetic analysis showed a biphasic behavior of the enzyme. Oxidized hydroxylamine and nitroso compounds in the reaction were detected both in vitro and in vivo based on LC–MS. The active site metal was confirmed to be iron based on a ferrozine assay. These findings provide new insights into the biosynthesis of chloramphenicol and could lead to further development of CmlI as a useful biocatalyst.     

Model studies of 6,7-indolequinone cofactors of quinoprotein amine dehydrogenases

The electronic effects of the C-4 substituent on the physicochemical properties and reactivity of the 6,7-inodolequinone cofactors (CTQ and TTQ) have extensively been investigated with use of a series of C-4 substituted 6,7-inodolequinone derivatives (1-4). The one-electron reduction potentials of the 6,7-inodolequinone derivatives decrease with increasing the electron donating ability of the C-4 substituent (with the following order of E degrees': 4>1>2>3). The reaction of indolequinones 1-3 with benzylamine proceeds stepwise through the iminoquinone and the product-imine intermediates to give aminophenol as the final product as the case of TTQ model compound 4. The rate constants of each step have been determined by the detailed kinetic analysis, and the kinetic deuterium isotope effects have also been examined to confirm the rate-determining step. The reactivity of CTQ model compound 1 toward the amines is by one order of magnitude lower than that of TTQ model compound 4. The reactivity of indolequinones 2 and 3 is further decreased due to their stronger electron-donating substituents at C-4. A more important difference between CTQ model compound 1 and TTQ model compound 4 is the reactivity of the iminoquinone intermediate: the reaction of the CTQ model compound with amines stops at the iminoquinone formation stage at room temperature, whereas the reaction of the TTQ model compound with amines proceeds up to the aminophenol formation. Thus, the energy barrier for the rearrangement of the iminoquinone to the product-imine is higher in the CTQ model system than in the TTQ model system.     

A top down approach to protein structural studies using chemical cross-linking and Fourier transform mass spectrometry

Mass spectrometric analysis of wild-type proteins that have been covalently modified by bifunctional cross-linking reagents and then digested proteolytically can be used to obtain low-resolution distance constraints, which can be useful for protein structure determination. Limitations of this approach include time-consuming separation steps, such as the separation of internally cross-linked protein monomers from covalent dimers, and a susceptibility to artifacts due to low levels of natural and man-made peptide modifications that can be mistaken for cross-linked species. The results presented here show that when a crude cross-linked protein mixture is injected into an electrospray ionization Fourier transform mass spectrometry (ESI-FTMS) instrument, the cross-link positions can be localized by fragmentation and mass spectrometry on the 'gas-phase purified' singly internally cross-linked monomer. Our results show that reaction of ubiquitin with the homobifunctional lysine-lysine cross-linking reagent dissuccinimidyl suberate (DSS) resulted in two cross-links consistent with the known ubiquitin tertiary structure (K6-K11 and K48-K63). Because no protein or peptide chemistry steps are needed, other than the initial cross-linking, this new top down approach appears well suited for high-throughput experiments with multiple cross-linkers and reaction conditions. Published in 2002 by John Wiley & Sons, Ltd.     

Transpeptidase-mediated incorporation of D-amino acids into bacterial peptidoglycan

The β-lactams are the most important class of antibiotics in clinical use. Their lethal targets are the transpeptidase domains of penicillin binding proteins (PBPs), which catalyze the cross-linking of bacterial peptidoglycan (PG) during cell wall synthesis. The transpeptidation reaction occurs in two steps, the first being formation of a covalent enzyme intermediate and the second involving attack of an amine on this intermediate. Here we use defined PG substrates to dissect the individual steps catalyzed by a purified E. coli transpeptidase. We demonstrate that this transpeptidase accepts a set of structurally diverse D-amino acid substrates and incorporates them into PG fragments. These results provide new information on donor and acceptor requirements as well as a mechanistic basis for previous observations that noncanonical D-amino acids can be introduced into the bacterial cell wall.     

Catalysis of Extracellular Deamination by a FAD-Linked Oxidoreductase after Prodrug Maturation in the Biosynthesis of Saframycin A

The biosynthesis of antibiotics in bacteria is usually believed to be an intracellular process, at the end of which the matured compounds are exported outside the cells. The biosynthesis of saframycin A (SFM-A), an antitumor antibiotic, requires a cryptic fatty acyl chain to guide the construction of a pentacyclic tetrahydroisoquinoline scaffold; however, the follow-up deacylation and deamination steps remain unknown. Herein we demonstrate that SfmE, a membrane-bound peptidase, hydrolyzes the fatty acyl chain to release the amino group; and SfmCy2, a secreted oxidoreductase covalently associated with FAD, subsequently performs an oxidative deamination extracellularly. These results not only fill in the missing steps of SFM-A biosynthesis, but also reveal that a FAD-binding oxidoreductase catalyzes an unexpected deamination reaction through an unconventional extracellular pathway in Streptmyces bacteria.     

Tunneling and classical paths for proton transfer in an enzyme reaction dominated by tunneling: oxidation of tryptamine by aromatic amine dehydrogenase

Proton tunneling dominates the oxidative deamination of tryptamine catalyzed by the enzyme aromatic amine dehydrogenase. For reaction with the fast substrate tryptamine, a H/D kinetic isotope effect (KIE) of 55 +/- 6 has been reported-one of the largest observed in an enzyme reaction. We present here a computational analysis of this proton-transfer reaction, applying combined quantum mechanics/molecular mechanics (QM/MM) methods (PM3-SRP//PM3/CHARMM22). In particular, we extend our previous computational study (Masgrau et al. Science 2006, 312, 237) by using improved energy corrections, high-level QM/MM methods, and an ensemble of paths to estimate the tunneling contributions. We have carried out QM/MM molecular dynamics simulations and variational transition state theory calculations with small-curvature tunneling corrections. The results provide detailed insight into the processes involved in the reaction. Transfer to the O2 oxygen of the catalytic base, Asp128beta, is found to be the favored reaction both thermodynamically and kinetically, even though O1 is closer in the reactant complex. Comparison of quantum and classical models of proton transfer allows estimation of the contribution of hydrogen tunneling in lowering the barrier to reaction in the enzyme. A reduction of the activation free energy due to tunneling of 3.1 kcal mol-1 is found, which represents a rate enhancement due to tunneling by 2 orders of magnitude. The calculated KIE of 30 is significantly elevated over the semiclassical limit, in agreement with the experimental observations; a semiclassical value of 6 is obtained when tunneling is omitted. A polarization of the C-H bond to be broken is observed due to the close proximity of the catalytic aspartate and the (formally) positively charged imine nitrogen. A comparison is also made with the related quinoprotein methylamine dehydrogenase (MADH)-the much lower KIE of 11 that we obtain for the MADH/methylamine system is found to arise from a more endothermic potential energy surface for the MADH reaction.     

Catalysis of Extracellular Deamination by a FAD‐Linked Oxidoreductase after Prodrug Maturation in the Biosynthesis of Saframycin A

The biosynthesis of antibiotics in bacteria is usually believed to be an intracellular process, at the end of which the matured compounds are exported outside the cells. The biosynthesis of saframycin A (SFM‐A), an antitumor antibiotic, requires a cryptic fatty acyl chain to guide the construction of a pentacyclic tetrahydroisoquinoline scaffold; however, the follow‐up deacylation and deamination steps remain unknown. Herein we demonstrate that SfmE, a membrane‐bound peptidase, hydrolyzes the fatty acyl chain to release the amino group; and SfmCy2, a secreted oxidoreductase covalently associated with FAD, subsequently performs an oxidative deamination extracellularly. These results not only fill in the missing steps of SFM‐A biosynthesis, but also reveal that a FAD‐binding oxidoreductase catalyzes an unexpected deamination reaction through an unconventional extracellular pathway in Streptmyces bacteria.     

Model Studies of TTQ-Containing Amine Dehydrogenases

The reactions of a TTQ model compound [1, 3-methyl-4-(3'-methylindol-2'-yl)indole-6,7-dione] with several amines have been investigated in organic media to obtain mechanistic information on the action of quinoprotein methylamine and aromatic amine dehydrogenases. It has been found that compound 1 acts as an efficient catalyst for the autorecycling oxidation of benzylamine by molecular oxygen in CH(3)OH. In order to evaluate the oxidation mechanism of amines by 1, the product analyses and kinetic studies have been carried out under anaerobic conditions. In the first stage of the reaction of 1 with amines, 1 is converted into an iminoquinone-type adduct (so-called substrateimine), which was isolated and characterized by using cyclopropylamine as a substrate. The observed NOE of the isolated product indicates clearly that the addition position of the amine is C-6 of the quinone. The molecular orbital calculations suggest that the thermodynamic stability of the carbinolamine intermediate is a major factor to determine such regioselectivity; the C-6 carbinolamine is more stable than the C-7 counterpart by 2.9 kcal/mol. The reactivity of several primary amines and the electronic effect of the p-substituents of benzylamine derivatives in the iminoquinone formation suggest that the addition step of the amine to the quinone is rate-determining. When amines having an acidic alpha-proton such as benzylamine derivatives are employed as substrates, formation of the iminoquinone adduct was followed by rearrangement to the productimine. The kinetic analysis has revealed that this rearrangement consists of noncatalyzed and general base-catalyzed processes. Large kinetic isotope effects of 7.8 and 9.2 were observed for both the noncatalyzed and general base-catalyzed processes, respectively, since these steps involve a proton abstraction from the alpha-position of the substrate. In the reaction with benzhydrylamine, the product imine was isolated quantitatively and well characterized by several spectroscopic data. In the case of benzylamine, the product imine is further converted into the aminophenol derivative by the imine exchange reaction with excess benzylamine. These results indicate clearly that the amine oxidation by compound 1 proceeds via a transamination mechanism as suggested for the enzymatic oxidation of amines by TTQ cofactor.     

Intra- and Inter-Molecular Cross-Linking of Peptide Ions in the Gas Phase: Reagents and Conditions

Intra-molecular and inter-molecular cross-linking of protonated polypeptide ions in the gas phase via ion/ion reactions have been demonstrated using N-hydroxysulfosuccinimide (sulfo-NHS)- based reagent anions. The initial step in the ion/ion reaction involves the formation of a long-lived complex between the peptide and reagent, which is a prerequisite for the covalent bioconjugation chemistry. The sulfonate groups on the NHS rings of the homo-bifunctional cross-linking reagents have high affinity for the protonated sites in the peptide and, therefore, facilitate the long-lived complex formation. In addition to the formation of a long-lived chemical complex, intra-molecular cross-linking also requires two unprotonated primary amine sites within a molecule where the covalent modification takes place. Alternatively, inter-molecular cross-linking demands the availability of one neutral primary amine site in each of the two peptides that are being cross-linked. Nucleophilic displacement of two sulfo-NHS groups by the amine functionalities in the peptide is a signature of the covalent cross-linking chemistry in the gas phase. Upon removal of the two sulfo-NHS groups, two amide bonds are formed between an unprotonated, primary amine group of a lysine side chain in the peptide and the carboxyl group in the reagent.     

Transannular Diels-Alder studies on the asymmetric total synthesis of chatancin: the pyranophane approach

[reaction: see text] A pathway is proposed for the biosynthesis of (+)-chatancin and (+)-sarcophytin linking these tetracycles to cembranoids by a pyranophane transannular Diels-Alder reaction. Preliminary synthetic results in this direction to reach macrocyclic dienedione 28 from farnesol are reported. Major synthetic steps include a Prins reaction, two enantioselective hydrogenations, and a macrocyclization via a beta-ketosulfoxide Michael-addition on an enone.     

Monitoring of the Sol-Gel Synthesis of Organic-inorganic Hybrids by FTIR Transmission,FTIR/ATR,NIR and Raman Spectroscopy

Summary: The sol-gel synthesis of organic-inorganic hybrids based on triethoxysilane- terminated poly(ethylene oxide) and tetraethylorthosilicate was monitored in-situ using three spectroscopic methods (FTIR/ATR, Raman, NIR). These spectroscopic methods allow in-situ monitoring of the evolution of hybrid materials starting from the modification of the polymer and the early steps of hydrolysis up to the network formation. By application of ²⁹Si solid-state NMR spectroscopy the assignment and quantification of the Raman bands to different end groups and different cross-linking states was made. The sol-gel reaction was also followed by in-line NIR spectroscopy. A multivariate data analysis was accomplished to obtain a conversion-time curve. Furthermore, we investigated spin-coated films on wafers using FTIR transmission spectroscopy.     

青霉素酰化酶交联酶聚体的制备及热失活动力学

以0.53 g/mL硫酸铵为沉淀剂, 0.35%(体积分数)戊二醛为交联剂制得青霉素酰化酶交联酶聚体(CLEAs), 酶活收率30.1%, 其最适温度(57 ℃)比游离酶提高10 ℃, 最适pH(10.0)向碱性偏移1.7个单位. 对比游离酶及其CLEAs的热稳定性和热失活动力学模型发现, 游离青霉素酰化酶制成CLEAs后, 其热失活动力学模型由一步失活转变为连串失活, 失活反应活化能由248.8 kJ/mol增加至549.2 kJ/mol, 对CLEAs热稳定性大幅提高的原因进行了解释. CLEAs重复利用7次后, 酶活保留56%以上, 具有良好的重复利用性.     

Analysis of the ambruticin and jerangolid gene clusters of Sorangium cellulosum reveals unusual mechanisms of polyketide biosynthesis

Ambruticins and jerangolids are structurally related antifungal polyketides produced by Sorangium cellulosum strains. Comparative analysis of the gene clusters and characterization of compounds produced by gene knockout strains suggested hypothetical schemes for biosynthesis of these compounds. Polyketide synthase (PKS) architecture suggests that the pyran ring structure common to ambruticins and jerangolids forms by an intramolecular reaction on a PKS-bound intermediate. Disrupting ambM, encoding a discrete enzyme homologous to PKS C-methyltransferase domains, gave 15-desmethylambruticins. Thus, AmbM is required for C-methylation, but not pyran ring formation. Several steps in the post-PKS modification of ambruticin involve new enzymology. Remarkably, the methylcyclopropane ring and putative carbon atom excision during ambruticin biosynthesis apparently occur on the PKS assembly line. The mechanism probably involves a Favorskii rearrangement, but further work is required to elucidate these complex events.     

Unprecedented Fe(IV) Species in a Diheme Protein MauG: A Quantum Chemical Investigation on the Unusual Mössbauer Spectroscopic Properties

Ferryl species are important catalytic intermediates in heme enzymes. A recent experimental investigation of a diheme protein MauG reported the first case of using two Fe(IV) species as an alternative to compound I in catalysis. Both Fe(IV) species have unusual M?ssbauer properties, which was found to originate from novel structural features based on a quantum chemical investigation. With comparison to the previously reported Fe(IV)=O and Fe(IV)-OH species, results here provide the first evidence of a couple of new mechanisms by which proteins influence the properties of ferryl species by directly providing the O via Tyr, or stabilizing exogenous O via hydrogen bonding interaction. These results expand our ability to identify and evaluate high-valent heme proteins and models.