Oct-2-yn-4-enoyl-CoA as a multifunctional enzyme inhibitor in fatty acid oxidation

Oct-2-yn-4-enoyl-CoA was found to be a multifunctional irreversible enzyme inhibitor in fatty acid oxidation mainly targeting mitochondrial trifunctional protein beta-subunit. It can also inactivate enoyl-CoA hydratase 2 and medium-chain acyl-CoA dehydrogenase. This study increased our understanding for the effect of acetylenic acids on fatty acid oxidation.     

Studies on the inactivation of bovine liver enoyl-CoA hydratase by (methylenecyclopropyl)formyl-CoA: elucidation of the inactivation mechanism and identification of cysteine-114 as the entrapped nucleophile

The inhibitory properties of (methylenecyclopropyl)formyl-CoA (MCPF-CoA), a metabolite derived from a natural amino acid, (methylenecyclopropyl)glycine, against bovine liver enoyl-CoA hydratase (ECH) were characterized. We have previously demonstrated that MCPF-CoA specifically targets ECHs, which catalyze the reversible hydration of alpha,beta-unsaturated enoyl-CoA substrates to the corresponding beta-hydroxyacyl-CoA products. Here, we synthesized (R)- and (S)-diastereomers of MCPF-CoA to examine the stereoselectivity of this inactivation. Both compounds were shown to be competent inhibitors for bovine liver ECH with nearly identical second-order inactivation rate constants (k(inact)/K(I)) and partition ratios (k(cat)/k(inact)), indicating that the inactivation is nonstereospecific with respect to ring cleavage. The inhibitor, upon incubation with bovine liver ECH, labels a tryptic peptide, ALGGGXEL, near the active site of the protein, where X is the amino acid that is covalently modified. Cloning and sequence analysis of bovine liver ECH gene revealed the identity of the amino acid residue entrapped by MCPF-CoA as Cys-114 (mature sequence numbering). On the basis of gHMQC (gradient heteronuclear multiple quantum coherence) analysis with [3-(13)C]-labeled MCPF-CoA, the ring cleavage is most likely induced by the nucleophilic attack at the terminal carbon of the exomethylene group (C(2)'). We propose a plausible inactivation mechanism that involves relief of ring strain and is consistent with examples found in the literature. In addition, these studies provide important clues for future design of more efficient and selective inhibitors to control and/or regulate fatty acid metabolism.     

Stereoselectivity of enoyl-CoA hydratase results from preferential activation of one of two bound substrate conformers

Enoyl-CoA hydratase catalyzes the hydration of trans-2-crotonyl-CoA to 3(S)- and 3(R)-hydroxybutyryl-CoA with a stereoselectivity (3(S)/3(R)) of 400,000 to 1. Importantly, Raman spectroscopy reveals that both the s-cis and s-trans conformers of the substrate analog hexadienoyl-CoA are bound to the enzyme, but that only the s-cis conformer is polarized. This selective polarization is an example of ground state strain, indicating the existence of catalytically relevant ground state destabilization arising from the selective complementarity of the enzyme toward the transition state rather than the ground state. Consequently, the stereoselectivity of the enzyme-catalyzed reaction results from the selective activation of one of two bound substrate conformers rather than from selective binding of a single conformer. These findings have important implications for inhibitor design and the role of ground state interactions in enzyme catalysis.     

Ring current effects in the active site of medium-chain Acyl-CoA dehydrogenase revealed by NMR spectroscopy

Medium-chain acyl-CoA dehydrogenase (MCAD) catalyzes the flavin-dependent oxidation of fatty acyl-CoAs to the corresponding trans-2-enoyl-CoAs. The interaction of hexadienoyl-CoA (HD-CoA), a product analogue, with recombinant pig MCAD (pMCAD) has been studied using (13)C NMR and (1)H-(13)C HSQC spectroscopy. Upon binding to oxidized pMCAD, the chemical shifts of the C1, C2, and C3 HD carbons are shifted upfield by 12.8, 2.1, and 13.8 ppm, respectively. In addition, the (1)H chemical shift of the C3-H is also shifted upfield by 1.31 ppm while the chemical shift of the C4 HD-CoA carbon is unchanged upon binding. These changes in chemical shift are unexpected given the results of previous Raman studies which revealed that the C3=C2-C1=O HD enone fragment is polarized upon binding to MCAD such that the electron density at the C3 and C1 carbons is reduced, not increased (Pellet et al. Biochemistry 2000, 39, 13982-13992). To investigate the apparent discrepancy between the NMR and Raman data for HD-CoA bound to MCAD, (13)C NMR spectra have been obtained for HD-CoA bound to enoyl-CoA hydratase, an enzyme system that has also previously been studied using Raman spectroscopy. Significantly, binding to enoyl-CoA hydratase causes the chemical shifts of the C1 and C3 HD carbons to move downfield by 4.8 and 5.6 ppm, respectively, while the C2 resonance moves upfield by 2.2 ppm, in close agreement with the alterations in electron density at these carbons predicted from Raman spectroscopy (Bell, A. F.; Wu, J.; Feng, Y.; Tonge, P. J. Biochemistry 2001, 40, 1725-33). The large increase in shielding experienced by the C1 and C3 HD carbons in the HD-CoA/MCAD complex is proposed to arise from the ring current field from the isoalloxazine portion of the flavin cofactor. The flavin ring current, which is only present when the enzyme is placed in an external magnetic field, also explains the differences in (13)C NMR chemical shifts for acetoacetyl-CoA when bound as an enolate to MCAD and enoyl-CoA hydratase and is used to rationalize the observation that the line widths of the C1 and C3 resonances are narrower when the ligands are bound to MCAD than when they are free in the protein solution.     

A specific gas chromatographic method for the determination of microsomal styrene monooxygenase and styrene epoxide hydratase activities.

A gas chromatographic (GC) method for the determination of the metabolite resulting from the activities of microsomal styrene monooxygenase (epoxide synthetase) and epoxide hydratase using styrene or styrene epoxide as substrates has been developed. The determination of the activities of both enzymes is based on the GC determination of phenylethylene glycol after its esterification with n-butylboronic acid. Kinetic parameters for both enzymes are given.     

Bioconversion of acrylonitruke to acrylamide using a thermostable nitrile hydratase

Although providing an attractive route for production of crrylamide from acrylonitrile, utilization of nitrile hydratase en zymes has been limited by the requirement for low temperatu rebioconversion conditions. Thisrreportsummarizes a search for thermostable nitrile hydratases from aerobic moderate thermophiles screened for ability to grow on acrylonitrileatt concentrations to 1% at elevated temperatures. A new isolate Bacillus sp. BR449 constitutively expresses a thermostble nitrile hydratase with properties iccluding low substrate inhibition and broad temperature range with optimal activity at 55°C. With prolonged exposure, BR449 nitrile hydratase exhibited temperature-dependent inactivation by acrylonitrile, which is attributed to alkylation of nucleophilic sites on the enzymes/     

Characteristics of peroxisome proliferation: co-induction of peroxisomal fatty acid oxidation-related enzymes with microsomal laurate hydroxylase

The profile of the changes in the peroxisomal fatty acid oxidation activity in rat liver was compared with that in microsomal omega-oxidation under various conditions such as a 2-week administration of phenoxyacetic acid derivatives and perfluorinated compounds, short and long-term administration of clofibrate and bezafibrate, high-fat diet feeding, starvation and diabetes. The results were summarized as follows: 1) when phenoxyacetic acid derivatives and perfluorinated compounds were administered, there was a significant correlation in the increase of the activities between peroxisomal fatty acid oxidation and microsomal omega-oxidation. 2) On the long-term administration (79 weeks) of peroxisome proliferators the activities of the enzymes were significantly reduced, but the levels were still higher than the control level in a similar manner. 3) On high-fat diet feeding the patterns of the changes in the activities of peroxisomal fatty acid oxidation, carnitine acetyltransferase and microsomal omega-oxidation were similar to each other, differing from the changes in the activities of microsomal aminopyrin demethylase and mitochondrial carnitine palmitoyltransferase. 4) Under starved and diabetic conditions, co-induction of peroxisomal fatty acid oxidation and microsomal omega-oxidation was observed. From these results it is suggested that 1) the biosynthesis of these enzymes would be regulated on the gene expression of the nearby domain and 2) peroxisomal fatty acid oxidation and microsomal omega-oxidation were co-operatively regulated in order to achieve fatty acid metabolism smoothly.     

Cobalt activation of Bacillus BR449 thermostable nitrile hydratase expressed in Escherichia coli

Expression of nitrile hydratase enzymes utilized in a new “green” process for acrylamide production has proven difficult in Escherichia coli owing to lack of a cobalt transport system to introduce the required cobaltion into this host. We describe the expression of a thermostable nitrile hydratase from a moderatethermophile Bacillus sp. BR449 in E. coli in which the cobaltrequired for enzyme activation is introduced by incubation, of the apoenzyme in the presence of Co⁺⁺ ion at 50°C, yielding active and thermostable, enzyme.     

The hydratase activity of malonate semialdehyde decarboxylase: mechanistic and evolutionary implications

Malonate semialdehyde decarboxylase (MSAD) is a member of the tautomerase superfamily, a group of structurally homologous proteins that have a characteristic beta-alpha-beta-fold and a catalytic amino-terminal proline. In addition to its physiological decarboxylase activity, the conversion of malonate semialdehyde to acetaldehyde and carbon dioxide, the enzyme has now been found to display a promiscuous hydratase activity, converting 2-oxo-3-pentynoate to acetopyruvate, with a kcat/Km value of 6.0 x 102 M-1 s-1. Pro-1 and Arg-75 are critical for both activities, and the pKa of Pro-1 was determined to be approximately 9.2 by a direct 15N NMR titration. These observations implicate a decarboxylation mechanism in which Pro-1 polarizes the carbonyl oxygen of substrate by hydrogen bonding and/or an electrostatic interaction. Arg-75 may position the carboxylate group into a favorable orientation for decarboxylation. Both the hydratase activity and the pKa value of Pro-1 are shared with trans-3-chloroacrylic acid dehalogenase, another tautomerase superfamily member that precedes MSAD in a bacterial degradation pathway for trans-1,3-dichloropropene. Hence, MSAD and CaaD could have evolved by divergent evolution from a common ancestral protein, retaining the necessary catalytic components for the conjugate addition of water.     

A novel inhibitor for Fe-type nitrile hydratase: 2-cyano-2-propyl hydroperoxide

Nitrile hydratase (NHase) is a non-heme iron or non-corrin cobalt enzyme having two post-translationally modified ligand residues, cysteine-sulfinic acid (alphaCys112-SO(2)H) and -sulfenic acid (alphaCys114-SOH). We studied the interaction between Fe-type NHase and isobutyronitrile (iso-BN) which had been reported as a competitive inhibitor with a K(i) value of 5 microM. From detailed kinetic studies of the inhibitory effect of iso-BN on Fe-type NHase, we found that authentic iso-BN was hydrated normally and that the impurity present in commercially available iso-BN inhibited NHase activity strongly. The inhibitory compound induced significant changes in the UV-vis absorption spectrum of NHase, suggesting its interaction with the iron center. This compound was purified by using reversed-phase HPLC and identified as 2-cyano-2-propyl hydroperoxide (Cpx) by (1)H and PFG-HMBC NMR spectroscopy. Upon addition of a stoichiometric amount of Cpx, NHase was irreversibly inactivated, probably by the oxidation of alphaCys114-SOH to Cys-SO(2)H. This result suggests that the -SOH structure of alphaCys114 is essential for the catalytic activity. The oxygen atom in Cys-SO(2)H is confirmed to come from the solvent H(2)O. The oxidized NHase was found to induce the UV-vis absorption spectral changes by addition of Cpx, suggesting that Cpx strongly interacted with iron(III) in the oxidized NHase to form a stable complex. Thus, Cpx functions as a novel irreversible inhibitor for NHase.     

Production and properties of 3-cyanopyridine hydratase inRhodococcus equi SHB-121

A strain ofRhodococcus equi SHB-121 forming 3-cyanopyridine hydratase was screened from nitrile-polluted soil. The optimum conditions for the formation of 3-cyanopyridine hydratase by the strain SHB-121 have been studied. Under the optimum conditions, the specific activity of the enzyme reached 5.32 U/mg of dry cell, 95 times higher than that cultured in screening medium. In addition, the activity of coexistent amidase was very low. 3-Cyanopyridine hydratase was purified from methylacrylamide-induced cells ofRh. equi SHB-121 by procedures including ultrasonic oscillation, ammonium sulfate precipitation, and column chromatographies on DEAE-cellulose DE52, hydroxyapatite, and Sephadex G-25. The overall purification was 31-fold. The molecular weight of the enzyme was about 30 kDA by SDS-PAGE. The pI value was 4.1. The transition temperature and pH were 7.0°C and 6.0, respectively, resulting from the differential spectra. The optimum pH and temperature for the enzyme reaction were 8.0 and 30°C. The enzyme activity was strongly inhibited by Ag⁺, Hg²⁺, Cu²⁺, and NH₄ ⁺, whereas it was enhanced by Fe³⁺ slightly. The enzyme catalyzed the hydration of 3-cyanopyridine to nicotinamide, and itsKm value was 0.1 mol/L. Uncompetitive inhibitor sodium cyanide has a K, value of 5 mmol/L.     

Enzyme–Substrate Binding Landscapes in the Process of Nitrile Biodegradation Mediated by Nitrile Hydratase and Amidase

The continuing discharge of nitriles in various industrial processes has caused serious environmental consequences of nitrile pollution. Microorganisms possess several nitrile-degrading pathways by direct interactions of nitriles with nitrile-degrading enzymes. However, these interactions are largely unknown and difficult to experimentally determine but important for interpretation of nitrile metabolisms and design of nitrile-degrading enzymes with better nitrile-converting activity. Here, we undertook a molecular modeling study of enzyme–substrate binding modes in the bi-enzyme pathway for degradation of nitrile to acid. Docking results showed that the top substrates having favorable interactions with nitrile hydratase from Rhodococcus erythropolis AJ270 (ReNHase), nitrile hydratase from Pseudonocardia thermophila JCM 3095 (PtNHase), and amidase from Rhodococcus sp. N-771 (RhAmidase) were benzonitrile, 3-cyanopyridine, and l-methioninamide, respectively. We further analyzed the interactional profiles of these top poses with corresponding enzymes, showing that specific residues within the enzyme’s binding pockets formed diverse contacts with substrates. This information on binding landscapes and interactional profiles is of great importance for the design of nitrile-degrading enzyme mutants with better oxidation activity toward nitriles or amides in the process of pollutant treatments.     

Effect of buffer general acid-base catalysis on the stereoselectivity of ester and thioester H/D exchange in D2O

As part of a comprehensive investigation on the stereochemistry of base-catalyzed 1,2-elimination and H/D exchange reactions of carbonyl compounds, we have found that the stereoselectivity of H/D exchange of 3-hydroxybutyryl N-acetylcysteamine (3) in D(2)O is strongly influenced by the presence of buffers. This buffer effect is also operative with a simple acyclic ester, ethyl 3-methoxybutanoate (7). Buffers whose general-acid components are cyclic tertiary ammonium ions are particularly effective in changing the stereoselectivity. (2)H NMR analysis showed that without buffer, H/D exchange of 3 produces 81-82% of the 2R*, 3R* diastereomer of 2-deuterio 3 (the anti product). In the presence of 0.33 M 3-quinuclidinone buffer, only 44% of the 2R*, 3R* diastereomer was formed. With ester 7, the stereoselectivity went from 93-94% in DO(-)/D(2)O to 60% in the presence of buffer. Phosphate buffer, as well as others, also showed substantial effects. The results are put into the context of what is known about the mechanism of H/D exchange of esters and thioesters, and the relevance of the buffer effect on the mechanism of the enoyl-CoA hydratase reaction is discussed. It is likely that hydrogen bonding in the enolate-buffer acid encounter complex is an important stereochemical determinant in producing a greater amount of the 2R*, 3S* diastereomer (the syn product). Studies that involve the protonation of enolate anions in D(2)O need to include the buffer general acid in any understanding of the stereoselectivity.     

Chiral Alcohols from Alkenes and Water: Directed Evolution of a Styrene Hydratase

Enantioselective synthesis of chiral alcohols through asymmetric addition of water across an unactivated alkene is a highly sought-after transformation and a big challenge in catalysis. Herein we report the identification and directed evolution of a fatty acid hydratase from Marinitoga hydrogenitolerans for the highly enantioselective hydration of styrenes to yield chiral 1-arylethanols. While directed evolution for styrene hydration was performed in the presence of heptanoic acid to mimic fatty acid binding, the engineered enzyme displayed remarkable asymmetric styrene hydration activity in the absence of the small molecule activator. The evolved styrene hydratase provided access to chiral alcohols from simple alkenes and water with high enantioselectivity (>99 : 1 e.r.) and could be applied on a preparative scale.     

The role of post-translational modification in the photoregulation of Fe-type nitrile hydratase

The inactive, nitrosyl bound form of Fe-type nitrile hydratase (NHase) contains two active site cysteine residues that are post-translationally modified to sulfenate (SO-) and sulfinate (SO2-) ligands. DFT and INDO/S calculations support the hypothesis that these unusual modifications play a key role in modulating the electronic absorption spectra and photoreactivity of the Fe(III) centre in the enzyme.     

Effects of <Emphasis Type="Italic">L</Emphasis>-Carnitine on Sucrose-Induced Hyperlipidaemia

Summary. L-Carnitine, L-(−)-β-hydroxy-γ-trimethylaminobutyrate, plays an important role as a factor necessary for the transport of long-chain fatty acids into the mitochondria. In order to investigate the influence of L-carnitine on hyperlipidaemias, the experimental model of the sucrose-induced hypertriglyceridaemia of the rat was used. In these experiments L-carnitine in the dose of 11 mg per day and 100 g body weight (over the period of 1 week) was able to antagonize the sucrose-induced hypertriglyceridaemia and the increase of serum free fatty acid level in female rats of the Wistar strain. Carnitine administration did not change the activities of lipogenic enzymes and fatty acid synthesis in the liver. However, L-carnitine increases the rate of hepatic fatty acid oxidation. Our results indicate a hypotriglyceridemic and free fatty acid lowering effect of L-carnitine, and suggest the use of this compound in the therapy of hyperlipidaemias.     

Effects of L-Carnitine on Sucrose-Induced Hyperlipidaemia

L-Carnitine, L-(−)-β-hydroxy-γ-trimethylaminobutyrate, plays an important role as a factor necessary for the transport of long-chain fatty acids into the mitochondria. In order to investigate the influence of L-carnitine on hyperlipidaemias, the experimental model of the sucrose-induced hypertriglyceridaemia of the rat was used. In these experiments L-carnitine in the dose of 11 mg per day and 100 g body weight (over the period of 1 week) was able to antagonize the sucrose-induced hypertriglyceridaemia and the increase of serum free fatty acid level in female rats of the Wistar strain. Carnitine administration did not change the activities of lipogenic enzymes and fatty acid synthesis in the liver. However, L-carnitine increases the rate of hepatic fatty acid oxidation. Our results indicate a hypotriglyceridemic and free fatty acid lowering effect of L-carnitine, and suggest the use of this compound in the therapy of hyperlipidaemias.     

Evolution of function in the crotonase superfamily: the stereochemical course of the reaction catalyzed by 2-ketocyclohexanecarboxyl-CoA hydrolase

Members of the mechanistically diverse enoyl-CoA hydratase (crotonase) superfamily catalyze reactions that involve stabilization of an enolate anion derived from an acyl thioester of coenzyme A. 2-Ketocyclohexanecarboxyl-CoA hydrolase (BadI), found in a pathway for anaerobic degradation of benzoate by Rhodopseudomonas palustris, is a member of the crotonase superfamily that catalyzes a reverse Dieckmann reaction in which the substrate is hydrolyzed to pimelyl-CoA. The substrate is the configurationally labile 2S-ketocyclohexanecarboxyl-CoA, and in 2H2O solvent hydrogen is incorporated into the 2-proS position of the pimelyl-CoA product. Therefore, the stereochemical course of the BadI-catalyzed reaction is inversion. This information is important for understanding the roles of active-site functional groups in the active site of BadI as well as in the active sites of the homologous 1,4-dihydroxynaphthoyl-CoA synthases that catalyze a forward Dieckmann reaction.     

Biotransformation of nitriles using the solvent-tolerant nitrile hydratase from Rhodopseudomonas palustris CGA009

A study has been carried out into the biocatalytic hydration of nitriles using the nitrile hydratase enzyme from Rhodopseudomonas palustris CGA009. It has been shown that this nitrile hydratase can hydrate aliphatic, aromatic and heterocyclic nitriles under very mild conditions, in mixtures of pH 7 buffer and a range of organic solvents, often with excellent chemoselectivity. The major determinant of hydration occurring is the degree of steric hindrance around the nitrile moiety and/or size of the substrates.     

Enzymatic hydrogen atom abstraction from polyunsaturated fatty acids

The oxidation of polyunsaturated fatty acids such as arachidonic and linoleic acid initiates a plethora of cell signaling pathways in animals and plants. The chemistry of the enzymatic oxidation has been investigated for several enzymes, most notably prostaglandin synthase and the lipoxygenases, revealing many surprises and impressive examples of enzymatic control of hydrogen atom abstraction and subsequent oxygenation.