Mechanism of enzymic isomerization and epimerization of D-erythrose 4-phosphate.
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ABSTRACT:
SUBMITTER: Hosomi S
PROVIDER: S-EPMC1147348 | biostudies-other | 1986 Nov
REPOSITORIES: biostudies-other
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Project description:Glucosamine 6-phosphate synthase converts fructose-6P into glucosamine-6P or glucose-6P depending on the presence or absence of glutamine. The isomerase activity is associated with a 40-kDa C-terminal domain, which has already been characterized crystallographically. Now the three-dimensional structures of the complexes with the reaction product glucose-6P and with the transition state analog 2-amino-2-deoxyglucitol-6P have been determined. Glucose-6P binds in a cyclic form whereas 2-amino-2-deoxyglucitol-6P is in an extended conformation. The information on ligand-protein interactions observed in the crystal structures together with the isotope exchange and site-directed mutagenesis data allow us to propose a mechanism of the isomerase activity of glucosamine-6P synthase. The sugar phosphate isomerization involves a ring opening step catalyzed by His504 and an enolization step with Glu488 catalyzing the hydrogen transfer from C1 to C2 of the substrate. The enediol intermediate is stabilized by a helix dipole and the epsilon-amino group of Lys603. Lys485 may play a role in deprotonating the hydroxyl O1 of the intermediate.
| S-EPMC2144271 | biostudies-other
Project description:There are five known general catalytic mechanisms used by enzymes to catalyze carbohydrate epimerization. The amino sugar epimerase N-acetylmannosamine-6-phosphate 2-epimerase (NanE) has been proposed to use a deprotonation-reprotonation mechanism, with an essential catalytic lysine required for both steps. However, the structural determinants of this mechanism are not clearly established. We characterized NanE from Staphylococcus aureus using a new coupled assay to monitor NanE catalysis in real time and found that it has kinetic constants comparable with other species. The crystal structure of NanE from Staphylococcus aureus, which comprises a triosephosphate isomerase barrel fold with an unusual dimeric architecture, was solved with both natural and modified substrates. Using these substrate-bound structures, we identified the following active-site residues lining the cleft at the C-terminal end of the β-strands: Gln11, Arg40, Lys63, Asp124, Glu180, and Arg208, which were individually substituted and assessed in relation to the mechanism. From this, we re-evaluated the central role of Glu180 in this mechanism alongside the catalytic lysine. We observed that the substrate is bound in a conformation that ideally positions the C5 hydroxyl group to be activated by Glu180 and donate a proton to the C2 carbon. Taken together, we propose that NanE uses a novel substrate-assisted proton displacement mechanism to invert the C2 stereocenter of N-acetylmannosamine-6-phosphate. Our data and mechanistic interpretation may be useful in the development of inhibitors of this enzyme or in enzyme engineering to produce biocatalysts capable of changing the stereochemistry of molecules that are not amenable to synthetic methods.
| S-EPMC8482478 | biostudies-literature
Project description:Post-translational modifications that do not result in a change in mass are particularly difficult to detect by mass spectrometry. For example, isomerization of aspartic acid or epimerization of any chiral residue within a peptide do not lead to mass shifts but can be identified by examination of independently acquired tandem mass spectra or by combination with another technique. For analysis of a biological sample, this means that liquid chromatography or some other type of separation must be used to first separate the isomers from one another. Furthermore, each specific m/z of interest must be sampled repeatedly to allow for comparison of the tandem mass spectra from each separated isomer, which contrasts with the traditional approach in proteomics where the goal is typically to avoid resampling the same m/z. We illustrate that isomerization and epimerization of peptides can be identified in this fashion by examination of long-lived crystallin proteins extracted from a sheep eye lens. Tandem mass spectrometry relying on a combination of radical directed dissociation (RDD) and collision induced dissociation (CID) following separation by liquid chromatography was used to identify modified peptides. Numerous sites of isomerization and epimerization, including several that have not been previously identified, were determined with peptide specificity. It is demonstrated that the specific sites of amino acid isomerization within each peptide can be identified by comparison with synthetic peptides. For ?-crystallin proteins, the sites that undergo the greatest degree of isomerization correspond to disordered regions, which may have important implications on chaperone functionality within the context of aging.
| S-EPMC4188265 | biostudies-literature
Project description:Aminoglycoside 2''-phosphotransferases are clinically important enzymes that cause high levels of resistance to aminoglycoside antibiotics by the organisms that harbor them. These enzymes phosphorylate aminoglycosides, and the modified antibiotics show significant reduction in the binding ability to target the bacterial ribosome. This report presents a detailed characterization of the antibiotic resistance profile and the aminoglycoside and nucleotide triphosphate substrate profiles of four common aminoglycoside 2''-phosphotransferases widely distributed in clinically important Gram-positive microorganisms. Although the antibiotic resistance phenotypes exhibited by these enzymes are similar, their aminoglycoside and nucleotide triphosphate substrate profiles are distinctive. Contrary to the dogma that these enzymes use ATP as the source of phosphate in their reactions, two of the four aminoglycoside 2'-phosphotransferases utilize GTP as the phosphate donor. Of the other two enzymes, one exhibits preference for ATP, and the other can utilize either ATP or GTP as nucleotide triphosphate substrate. A new nomenclature for these enzymes is put forth that takes into account the differences among these enzymes based on their respective substrate preferences. These nucleotide triphosphate preferences should have ramifications for understanding of the evolution, selection, and dissemination of the genes for these important resistance enzymes.
| S-EPMC2652286 | biostudies-literature
Project description:The catalytic antibody 4B2, which was generated against a substituted amidine 1, catalyses the allylic isomerization of beta, gamma-unsaturated ketones with an acceleration factor (k(cat)/k(uncat)) of 1.5x10(3). On the basis of the 'bait and switch' strategy, it was reasoned that the positively charged hapten could elicit, by charge complementarity, an acidic residue (Asp or Glu) in the antibody-binding site in the right position to catalyse this proton transfer reaction. The pH dependence curve of k(cat)/K(m) shows a bell-shaped feature with an optimum at approx. pH 4.5. By cloning and sequencing the light and heavy chains of the 4B2 antibody, we confirmed the presence of several Asp and Glu residues in the complementarity-determining region loops. The antibody catalyses the alpha-proton exchange on the same substrates, demonstrating the involvement of a dienol intermediate in the reaction mechanism. Kinetic studies with (2)H-NMR provide evidence that alpha-proton abstraction is stereospecific. Whether the process involves one or two acid/base residues in this simple proton transfer or whether it is a concerted mechanism is discussed.
| S-EPMC1220901 | biostudies-other
Project description:Sugar 4-epimerization reactions are important for the production of rare sugars and their derivatives, which have various potential industrial applications. For example, the production of tagatose, a functional sweetener, from fructose by sugar 4-epimerization is currently constrained because a fructose 4-epimerase does not exist in nature. We found that class II D-fructose-1,6-bisphosphate aldolase (FbaA) catalyzed the 4-epimerization of D-fructose-6-phosphate (F6P) to D-tagatose-6-phosphate (T6P) based on the prediction via structural comparisons with epimerase and molecular docking and the identification of the condensed products of C3 sugars. In vivo, the 4-epimerization activity of FbaA is normally repressed. This can be explained by our results showing the catalytic efficiency of D-fructose-6-phosphate kinase for F6P phosphorylation was significantly higher than that of FbaA for F6P epimerization. Here, we identified the epimerization reactions and the responsible catalytic residues through observation of the reactions of FbaA and L-rhamnulose-1-phosphate aldolases (RhaD) variants with substituted catalytic residues using different substrates. Moreover, we obtained detailed potential epimerization reaction mechanism of FbaA and a general epimerization mechanism of the class II aldolases L-fuculose-1-phosphate aldolase, RhaD, and FbaA. Thus, class II aldolases can be used as 4-epimerases for the stereo-selective synthesis of valuable carbohydrates.
| S-EPMC5434028 | biostudies-literature
Project description:In the biosynthesis of heparin and heparan sulphate, D-glucuronic acid residues are converted into L-iduronic acid (IdoA) units by C-5 epimerization, at the polymer level. The reaction catalysed by the epimerase occurs by reversible abstraction and readdition of a proton at C-5 of target hexuronic acid residues, through a carbanion intermediate, with or without an inversion of configuration at C-5 [Prihar, Campbell, Feingold, Jacobsson, Jensen, Lindahl and Rodén (1980) Biochemistry 19, 495-500]. Incubation of chemically N-sulphated capsular polysaccharide from Escherichia coli K5 ([4GlcAbeta1-4GlcNSO(3)alpha1-](n)), or of O-desulphated heparin (predominantly [4IdoAalpha1-4GlcNSO(3)alpha1-](n)) with purified C-5 epimerase from bovine liver, resulted in the interconversion of glucuronic acid and IdoA residues, which reached equilibrium (30-40% IdoA/total hexuronic acid) after approx. 1 h of incubation. Similar incubations performed in the presence of (3)H(2)O resulted in progressive labelling at C-5 of the target hexuronic acid units of either substrate polysaccharide. Contrary to chemical D-gluco/L-ido equilibrium, established within 1 h of incubation, the accumulation of (3)H label continued for at least 6 h. This isotope effect suggests that the second stage of the reaction, i.e. the re-addition of a proton to the carbanion intermediate, is the rate-limiting step of the overall process. Analysis of the 5-(3)H-labelled polysaccharide products showed that the (3)H was approximately equally distributed between glucuronic acid and IdoA units, irrespective of incubation time (from 15 min to 72 h) and of the relative proportions of the two epimers in the substrate. This finding points to a catalytic mechanism in which the abstraction and re-addition of C-5 protons are effected by two polyprotic bases, presumably lysine residues. Previous experiments relating to the biosynthesis of dermatan sulphate were similarly interpreted in terms of a two-base epimerization mechanism but differed from the present findings by implicating one monoprotic and one polyprotic base function [Hannesson, Hagner-McWhirter, Tiedemann, Lindahl and Malmström (1996) Biochem. J. 313, 589-596].
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Project description:The polyketide synthases responsible for the biosynthesis of the polyether antibiotics nanchangmycin (1) and salinomycin (4) harbor a number of redox-inactive ketoreductase (KR0) domains that are implicated in the generation of C2-epimerized (2S)-2-methyl-3-ketoacyl-ACP intermediates. Evidence that the natural substrate for the polyether KR0 domains is, as predicted, a (2R)-2-methyl-3-ketoacyl-ACP intermediate, came from a newly developed coupled ketosynthase (KS)-ketoreductase (KR) assay that established that the decarboxylative condensation of methylmalonyl-CoA with S-propionyl-N-acetylcysteamine catalyzed by the Nan[KS1][AT1] didomain from module 1 of the nanchangmycin synthase generates exclusively the corresponding (2R)-2-methyl-3-ketopentanoyl-ACP (7a) product. In tandem equilibrium isotope exchange experiments, incubation of [2-2H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-ACP (6a) with redox-active, epimerase-inactive EryKR6 from module 6 of the 6-deoxyerythronolide B synthase and catalytic quantities of NADP+ in the presence of redox-inactive, recombinant NanKR10 or NanKR50, from modules 1 and 5 of the nanchangmycin synthase, or recombinant SalKR70 from module 7 of the salinomycin synthase, resulted in first-order, time-dependent washout of deuterium from 6a. Control experiments confirmed that this washout was due to KR0-catalyzed isotope exchange of the reversibly generated, transiently formed oxidation product [2-2H]-(2R)-2-methyl-3-ketopentanoyl-ACP (7a), consistent with the proposed epimerase activity of each of the KR0 domains. Although they belong to the superfamily of short chain dehydrogenase-reductases, the epimerase-active KR0 domains from polyether synthases lack one or both residues of the conserved Tyr-Ser dyad that has previously been implicated in KR-catalyzed epimerizations.
| S-EPMC5332327 | biostudies-literature
Project description:Vertebrate rhodopsin (Rh) contains 11-cis-retinal as a chromophore to convert light energy into visual signals. On absorption of light, 11-cis-retinal is isomerized to all-trans-retinal, constituting a one-way reaction that activates transducin (Gt) followed by chromophore release. Here we report that bovine Rh, regenerated instead with a six-carbon-ring retinal chromophore featuring a C11=C12 double bond locked in its cis conformation (Rh6mr), employs an atypical isomerization mechanism by converting 11-cis to an 11,13-dicis configuration for prolonged Gt activation. Time-dependent UV-vis spectroscopy, HPLC, and molecular mechanics analyses revealed an atypical thermal reisomerization of the 11,13-dicis to the 11-cis configuration on a slow timescale, which enables Rh6mr to function in a photocyclic manner similar to that of microbial Rhs. With this photocyclic behavior, Rh6mr repeatedly recruits and activates Gt in response to light stimuli, making it an excellent candidate for optogenetic tools based on retinal analog-bound vertebrate Rhs. Overall, these comprehensive structure-function studies unveil a unique photocyclic mechanism of Rh activation by an 11-cis-to-11,13-dicis isomerization.
| S-EPMC5380078 | biostudies-literature
Project description:Proline isomerization is a ubiquitous process that plays a key role in the folding of proteins and in the regulation of their functions. Different families of enzymes, known as "peptidyl-prolyl isomerases" (PPIases), catalyze this reaction, which involves the interconversion between the cis and trans isomers of the N-terminal amide bond of the amino acid proline. However, complete descriptions of the mechanisms by which these enzymes function have remained elusive. We show here that cyclophilin A, one of the most common PPIases, provides a catalytic environment that acts on the substrate through an electrostatic handle mechanism. In this mechanism, the electrostatic field in the catalytic site turns the electric dipole associated with the carbonyl group of the amino acid preceding the proline in the substrate, thus causing the rotation of the peptide bond between the two residues. We identified this mechanism using a combination of NMR measurements, molecular dynamics simulations, and density functional theory calculations to simultaneously determine the cis-bound and trans-bound conformations of cyclophilin A and its substrate as the enzymatic reaction takes place. We anticipate that this approach will be helpful in elucidating whether the electrostatic handle mechanism that we describe here is common to other PPIases and, more generally, in characterizing other enzymatic processes.
| S-EPMC4104850 | biostudies-literature