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At least 1,135 records · Page 63Linked to original sources
PubMed2006

Mechanism of dihydroneopterin aldolase: functional roles of the conserved active site glutamate and lysine residues.

Dihydroneopterin aldolase (DHNA) catalyzes the conversion of 7,8-dihydroneopterin (DHNP) to 6-hydroxymethyl-7,8-dihydropterin (HP) in the folate biosynthetic pathway. There are four conserved active site residues at the active site, E22, Y54, E74, and K100 in Staphylococcus aureus DHNA (SaDHNA), corresponding to E21, Y53, E73, and K98, respectively, in Escherichia coli DHNA (EcDHNA). The functional roles of the conserved glutamate and lysine residues have been investigated by site-directed mutagenesis in this work. E22 and E74 of SaDHNA and E21, E73, and K98 of EcDHNA were replaced with alanine. K100 of SaDHNA was replaced with alanine and glutamine. The mutant proteins were characterized by equilibrium binding, stopped-flow binding, and steady-state kinetic analyses. For SaDHNA, none of the mutations except E74A caused dramatic changes in the affinities of the enzyme for the substrate or product analogues or the rate constants. The Kd values for SaE74A were estimated to be >3000 microM, suggesting that the Kd values of the mutant are at least 100 times those of the wild-type enzyme. For EcDHNA, the E73A mutation increased the Kd values for the substrate or product analogues neopterin (MP), monapterin (NP), and 6-hydroxypterin (HPO) by factors of 340, 160, and 5600, respectively, relative to those of the wild-type enzyme. The K98A mutation increased the Kd values for NP, MP, and HPO by factors of 14, 3.6, and 230, respectively. The E21A mutation increased the Kd values for NP and HPO by factors of 2.2 and 42, respectively, but decreased the Kd value for MP by a factor of 3.3. The E22 (E21) and K100 (K98) mutations decreased the kcat values by factors of 1.3-2 x 10(4). The E74 (E73) mutation decreased in the kcat values by factors of approximately 10. The results suggested that E74 of SaDHNA and E73 of EcDHNA are important for substrate binding, but their roles in catalysis are minor. In contrast, E22 and K100 of SaDHNA are important for catalysis, but their roles in substrate binding are minor. On the other hand, E21 and K98 of EcDHNA are important for both substrate binding and catalysis.

Aldehyde-Lyases↗
PubMed2006

A point mutation converts dihydroneopterin aldolase to a cofactor-independent oxygenase.

Dihydroneopterin aldolase (DHNA) catalyzes the conversion of 7,8-dihydroneopterin (1) to 6-hydroxymethyl-7,8-dihydropterin (4) in the folate biosynthetic pathway. Substitution of a conserved tyrosine residue at the active site of DHNA by phenylalanine converts the enzyme to a cofactor-independent oxygenase, which generates mainly 7,8-dihydroxanthopterin (6) rather than 4. 6 is generated via the same enol intermediate as in the wild-type enzyme-catalyzed reaction, but this species undergoes an oxygenation reaction to form 6. The conserved tyrosine residue plays only a minor role in the formation of the enol reaction intermediate but a critical role in the protonation of the enol intermediate to form 4.

Aldehyde-Lyases↗
PubMed2004

Quantitative relationship between trimethylamine oxide aldolase activity and formaldehyde accumulation in white muscle from gadiform fish during frozen storage.

The accumulation of formaldehyde and the resulting deterioration of seafood products during frozen storage are primarily caused by the enzymatic activity of trimethylamine oxide aldolase (TMAOase). A screening of muscle samples from 24 species showed TMAOase activity in only the nine gadiform species that were analyzed. Enzyme activities in the major white muscle of gadiform fish showed large variations between species as well as between individuals. A frozen storage experiment showed a similarly large variation in the rate of formaldehyde accumulation, which could be accounted for by the endogenous white muscle in situ TMAOase activity. This TMAOase activity also correlated with the rate of insolubilization of otherwise high ionic strength soluble protein. A simple model describing the accumulation of free formaldehyde during frozen storage of gadiform fish is proposed. The model is based on a storage time-dependent decay of substrate-saturated white muscle TMAOase activity.

Aldehyde-Lyases↗
PubMed2004

Discovery of potent inhibitors of dihydroneopterin aldolase using CrystaLEAD high-throughput X-ray crystallographic screening and structure-directed lead optimization.

Potent inhibitors of 7,8-dihydroneopterin aldolase (DHNA; EC 4.1.2.25) have been discovered using CrystaLEAD X-ray crystallographic high-throughput screening followed by structure-directed optimization. Screening of a 10 000 compound random library provided several low affinity leads and their corresponding X-ray crystal structures bound to the enzyme. The presence of a common structural feature in each of the leads suggested a strategy for the construction of a directed library of approximately 1000 compounds that were screened for inhibitory activity in a traditional enzyme assay. Several lead compounds with IC(50) values of about 1 microM against DHNA were identified, and crystal structures of their enzyme-bound complexes were obtained by cocrystallization. Structure-directed optimization of one of the leads thus identified afforded potent inhibitors with submicromolar IC(50) values.

Aldehyde-Lyases↗
PubMed2003

One-pot synthesis of L-Fructose using coupled multienzyme systems based on rhamnulose-1-phosphate aldolase.

Two methods have been developed for the highly efficient enzymatic synthesis of L-fructose: one is based on rhamnulose-1-phosphate aldolase and acid phosphatase using racemic glyceraldehyde and dihydroxyacetone phosphate as substrates; the other is to generate enantiomerically pure L-glyceraldehyde in situ from glycerol for the aldol reaction, using galactose oxidase catalyzed oxidation of glycerol in the presence of catalase. Using this four-enzyme system, enantiomerically pure L-fructose was obtained. Using the more expensive dihydroxyacetone phosphate, the yield was 55% after purification.

Acid Phosphatase↗
PubMed2006

A class II aldolase mimic.

[structures: see text] A class II aldolase-mimicking synthetic polymer was prepared by the molecular imprinting of a complex of cobalt (II) ion and either (1S,3S,4S)-3-benzoyl-1,7,7-trimethylbicyclo[2.2.1]heptan-2-one (4a) or (1R,3R,4R)-3-benzoyl-1,7,7-trimethylbicyclo[2.2.1]heptan-2-one (4b) in a 4-vinylpyridine-styrene-divinylbenzene copolymer. Evidence for the formation of interactions between the functional monomer and the template was obtained from NMR and VIS titration studies. The polymers imprinted with the template demonstrated enantioselective recognition of the corresponding template structure, and induced a 55-fold enhancement of the rate of reaction of camphor (1) with benzaldehyde (2), relative to the solution reactions, and were also compared to reactions with a series of reference polymers. Substrate chirality was observed to influence reaction rate, and the reaction could be competitively inhibited by dibenzoylmethane (6). Collectively, the results presented provide the first example of the use of enantioselective molecularly imprinted polymers for the catalysis of carbon-carbon bond formation.

Aldehyde-Lyases↗
PubMed1999

Substrate-Assisted Catalysis in Sialic Acid Aldolase.

Sialic acid aldolase catalyses the reversible aldol condensation of pyruvate and N-acetylmannosamine with an apparent lack of stereospecificity. Consistent with this, modeling of Schiff base and enamine intermediates in the active site of this enzyme yields two conformations, corresponding to si- and re-face attack in the aldol condensation reaction. The acceptor-aldehyde group is found on different sides of the enamine in the two conformations, but with the remainder of the substrate having very similar geometries in the protein. No histidine residue previously speculated to function as a general base in the mechanism is found near the enzyme active site. In the absence of functionally active groups in the active site, the carboxylate of the substrate is proposed to function as the general acid/base. Molecular orbital calculations indicate that the barrier to aldol cleavage via this mechanism in the gas phase of the related system, 4-hydroxy-2-methyiminopentanoic acid, is 74 kJ mol(-)(1).

Journal Article↗
PubMed2006

Mechanism of dihydroneopterin aldolase: a molecular dynamics study of the apo enzyme and its product complex.

Dihydroneopterin aldolase (DHNA), an enzyme in the pathway that generates folic acid in bacteria, is investigated by a series of molecular dynamics simulations in its free form and complexed with its product, 6-hydroxymethyl-7,8-dihydropterin (HP). The active sites in DHNA are formed at the interface between pairs of protomers in this octameric protein. On the basis of root-mean-square deviation and root-mean-square fluctuation analyses of the trajectories, which take advantage of the presence of eight active sites, flexible regions of the apo protein surrounding the active site are identified and, upon binding HP, show that the active site is rigidified. Specific residues, associated with binding and the catalytic mechanism of DHNA, are associated with these flexible regions, and their interactions with HP account for most of the binding energy. A Principal Component Analysis shows rigidification of DHNA upon HP binding and that only a few modes of motion capture most of the atomic fluctuations in both apo and HP-bound forms. HP is pushed out of the active site in a series of simulations with different restrained positions between HP and DHNA to obtain a view of the exit pathway and energetic barrier to product release. The chosen pathway leads to a minimal disturbance of the system and provides a barrier consistent with the experimentally determined rate of product release. An analysis of the various components that contribute to the exit path energy and entropy provides insight into the energy-entropy compensation for product release.

Aldehyde-Lyases↗
PubMed2005

Mimicking dihydroxy acetone phosphate-utilizing aldolases through organocatalysis: a facile route to carbohydrates and aminosugars.

[reaction: see text] A practical and environmentally friendly organocatalytic strategy designed to mimic the DHAP aldolases has been developed and shown to be effective in the preparation of carbohydrates and aminosugars. (S)-Proline and (S)-2-pyrrolidine-tetrazole catalyzed the aldol reaction between dihydroxy acetone variants such as 1,3-dioxan-5-one and 2,2-dimethyl-1,3-dioxan-5-one with aldehydes to give the corresponding polyols in good yields with very high ees.

Aldehyde-Lyases↗
PubMed2006

Aldolase-catalyzed synthesis of beta-D-galp-(1-->9)-D-KDN: a novel acceptor for sialyltransferases.

[reaction: see text] beta-D-Galp-(1-->9)-D-KDN, a disaccharide component of the cell wall of Streptomyces sp. MB-8, was synthesized from beta-D-Galp-(1-->6)-D-Manp and pyruvate using a sialic acid aldolase. The obtained KDN-containing compound was a novel acceptor for bacterial sialyltransferases. Unusual alpha2,3- and alpha2,6-linked sialyltrisaccharides and a tetrasaccharide were synthesized using a one-pot two-enzyme system containing a Neisseria meningitidis CMP-sialic acid synthetase and a Pasteurella multocida sialyltransferase or a Photobacterium damsela alpha2,6-sialyltransferase.

Catalysis↗
PubMed2006

A novel adaptation of aldolase regulates virulence in Streptococcus pyogenes.

Regulation of virulence factor expression is critical for pathogenic microorganisms that must sense and adapt to a dynamic host environment; yet, the signal transduction pathways that enable this process are generally poorly understood. Here, we identify LacD.1 as a global regulator of virulence factor expression in the versatile human pathogen, Streptococcus pyogenes. LacD.1 is derived from a class I tagatose-1,6-bisphosphate aldolase homologous to those involved in lactose and galactose metabolism in related prokaryotes. However, regulation of transcription by LacD.1 is not dependent on this enzymatic activity or the canonical catabolite repression pathway, but likely does require substrate recognition. Our results suggest that LacD.1 has been adapted as a metabolic sensor, and raise the possibility that regulation of gene expression by metabolic enzymes may be a novel mechanism by which Gram-positive bacteria, including S. pyogenes, coordinate multiple environmental cues, allowing essential transcription programs to be coupled with perceived nutritional status.

Aldehyde-Lyases↗
PubMed2003

Theozyme for antibody aldolases. Characterization of the transition-state analogue.

A theozyme for antibody aldolases has been studied at the MP2/6-31G** computational level. Formation of two cooperative hydrogen-bonds between the acidic hydrogen atoms of the enamine and of a methanol molecule with the oxygen atom of the aldol acceptor markedly favors the C-C bond-formation associated with the aldol reaction. A comparative analysis of the geometry, the charge distribution and the shape of the molecular electrostatic potential of the transition structure (TS) with the covalent adduct, resulting from the reaction of methylamine and the beta-diketone used as a hapten allows us to characterize the transition-state analogue (TSA) generated at immunization. This finding allows us to propose a hapten based on a chiral beta-ketosulfoxide that could give the formation of a TSA that addresses the tetrahedral geometry of the TS.

Aldehyde-Lyases↗
PubMed2005

Synthesis of screening substrates for the directed evolution of sialic acid aldolase: towards tailored enzymes for the preparation of influenza A sialidase inhibitor analogues.

The stereoselective synthesis of two epimeric screening substrates, (4R, 5R, 6R)- and (4S, 5R, 6R)-6-dipropylcarbamoyl-2-oxo-4,5,6-trihydroxy-hexanoic acid, for the directed evolution of sialic acid aldolase is described. The complementary methods relied on stereoselective indium-mediated additions of ethyl alpha-bromomethyl acrylate to functionalised aldehydes. With an alpha-hydroxy aldehyde, (2R, 3R)-2,3-dihydroxy-4-oxo butanoic acid dipropylamide, the addition was chelation controlled, and the syn product, (6R, 5R, 4S)-6-dipropylcarbamoyl-2-methylidene-4,5,6-trihydroxy-hexanoic acid ethyl ester, was obtained. In contrast, the stereochemical outcome of the addition to (2R, 3R)-N,N-dipropyl-2,3-O-isopropylidene-4-oxobutyramide was consistent with Felkin-Anh control, and the anti adduct, (4R, 5R, 6R)-6-dipropylcarbamoyl-2-methylidene-4-hydroxy-5,6-O-isopropylidene-hexanoic acid ethyl ester, was the major product. Ozonolysis and deprotection gave the screening substrates as mixtures of furanose and pyranose forms, in good yields.

Directed Molecular Evolution↗
PubMed2005

'Top-down' characterization of site-directed mutagenesis products of Staphylococcus aureus dihydroneopterin aldolase by multistage tandem mass spectrometry in a linear quadrupole ion trap.

The gas-phase fragmentation reactions of a series of site-directed mutagenesis products of Staphylococcus aureus dihydroneopterin aldolase have been examined by multistage tandem mass spectrometry (MS/MS and MS(3)) in a linear quadrupole ion trap in order to explore the utility of this instrumentation for routine 'top-down' recombinant protein characterization. Following a rapid low resolution survey of the fragmentation behavior of the precursor ions from the wild type (WT) protein, selected charge states were subjected to detailed structural characterization by using high resolution 'zoom' and 'ultrazoom' resonance ejection MS/MS product ion scans. Dissociation of the [M + 18H](18+) charge state yielded a range of product ions from which extensive sequence information could be derived. In contrast, dissociation of the [M + 20H](20+) charge state resulted in a single dominant y(96) product ion formed by fragmentation between adjacent Ile/Gly residues, with only limited sequence coverage. Further extensive sequence information was readily obtained however, by MS(3) dissociation of this initial product. From the combined MS/MS and MS(3) spectra an overall sequence coverage of 66.9%, with fragmentation of 85 of the 127 amide bonds within the WT protein, was obtained. MS/MS and MS(3) of three of the four site-directed mutagenesis products (E29A), (Y61F) and (E81A) were found to yield essentially identical product ion spectra to the WT protein, indicating that these modifications had no significant influence on the fragmentation behavior. The specific site of modification could be unambiguously determined in each case by characterization of product ions resulting from fragmentation of amide bonds on either side of the mutation site. In contrast, MS/MS and MS(3) of the K107A mutant led to significantly different product ion spectra dominated by cleavages occurring N-terminal to proline, which restricted the ability to localize the modification site to within only an 8 amino acid region of the sequence. This work highlights the need for further studies to characterize the charge state, sequence and structural dependence to the low energy collision induced dissociation reactions of multiply protonated intact protein ions.

Aldehyde-Lyases↗
PubMed1970

Purification and properties of threonine aldolase from Clostridium pasteurianum.

1. Threonine aldolase was purified about 200-fold in 10% yield from Clostridium pasteurianum and its properties were examined. The final preparation gave three bands after ionophoresis on polyacrylamide gel. 2. The purified enzyme was shown to produce glycine and acetaldehyde in stoicheiometric amounts from threonine. The reverse reaction was demonstrated qualitatively. 3. The enzyme has a broad pH optimum at 6.5-7.0. 4. The enzyme is highly specific for l-threonine. 5. The enzyme is completely inhibited by 1mm concentrations of hydroxylamine and semicarbazide. Activity is decreased to 20% of the original by treatment with cysteine plus mercaptoethanol; most of the loss is regained on incubation with pyridoxal phosphate. It is concluded that pyridoxal phosphate is a prosthetic group. 6. The relationship between velocity and substrate concentration is atypical but indicates a K(m) value of 0.42mm. 7. The enzyme was demonstrated in several other strictly anaerobic bacteria.

Aldehyde-Lyases↗
PubMed1971

Studies on N-acetylneuraminic acid aldolase.

N-Acetylneuraminic acid aldolase from Clostridium perfringens was irreversibly inactivated by 1mm-bromopyruvate with a half-life of 4.2min at pH7.2 and 37 degrees C. The rate of inactivation was diminished in the presence of pyruvate but not with N-acetyl-d-mannosamine, indicating that the inhibitor acted at, or close to, the pyruvate-binding site. The apparent K(i) for bromopyruvate, calculated from the variation of half-life with inhibitor concentration, was 0.46mm, compared with a competitive K(i) 3.0mm for pyruvate. Incubation of the enzyme with radioactive bromopyruvate gave a radioactive, enzymically inactive, protein in which the bromopyruvate had alkylated cysteine residues. Incubation of the enzyme with radioactive pyruvate, followed by reduction with sodium borohydride, led to inactivation of the enzyme and binding of the pyruvate to the protein by reduction of a Schiff's base initially formed with the in-amino group of a lysine residue; only one-twentieth as many pyruvyl residues were bound by this method, showing that bromopyruvate is not specific for the active site. After protection of the enzyme active site with pyruvate, treatment with unlabelled bromopyruvate and dialysis, the enzyme retained 72% activity. When this treated enzyme was separately incubated with radioactive bromopyruvate, or radioactive pyruvate followed by sodium borohydride, the ratio of radioactive pyruvyl residues bound by the two methods was 2.3:1. After reduction and hydrolysis of the bromopyruvate-treated enzyme, the only detectable radioactive amino acid derivative was chromatographically and electrophoretically identical with S-(3-lactic acid)-cysteine. The enzyme was fully active in the presence of EDTA and was not stimulated by bivalent metal ions. It was strongly inhibited by silver and mercuric ions. The apparent molecular weight, determined by Sephadex chromatography, was 250000. A mechanism of action is proposed for the enzyme. Bromopyruvate reacts rapidly at pH6.0 with thiol-containing amino acids. Cysteine appears to react anomalously.

Binding Sites↗