Construction of a multifunctional enzyme complex via the strain-promoted azide-alkyne cycloaddition.

Inspired by the multienzyme complexes occurring in nature, enzymes have been brought together in vitro as well. We report a co-localization strategy milder than nonspecific cross-linking, and free of any scaffold and affinity tags. Using non-natural amino acid incorporation, two heterobifunctional linkers, and the strain-promoted azide-alkyne cycloaddition as conjugation reaction, three metabolic enzymes are linked together in a controlled manner. Conjugate formation was demonstrated by size-exclusion chromatography and gel electrophoresis. The multienzyme complexes were further characterized by native mass spectrometry. It was shown that the complexes catalyzed the three-step biosynthesis of piceid in vitro with comparable kinetic behavior to the uncoupled enzymes. The approach is envisioned to have high potential for various biotechnological applications, in which multiple biocatalysts collaborate at low concentrations, in which diffusion may be limited and/or side-reactions are prone to occur.

Stable isotope labeling by essential nutrients in cell culture for preparation of labeled coenzyme A and its thioesters.

Stable isotope dilution mass spectrometry (MS) represents the gold standard for quantification of endogenously formed cellular metabolites. Although coenzyme A (CoA) and acyl-CoA thioester derivatives are central players in numerous metabolic pathways, the lack of a commercially available isotopically labeled CoA limits the development of rigorous MS-based methods. In this study, we adapted stable isotope labeling by amino acids in cell culture (SILAC) methodology to biosynthetically generate stable isotope labeled CoA and thioester analogues for use as internal standards in liquid chromatography/multiple reaction monitoring mass spectrometry (LC/MRM-MS) assays. This was accomplished by incubating murine hepatocytes (Hepa 1c1c7) in media in which pantothenate (a precursor of CoA) was replaced with [(13)C(3)(15)N(1)]-pantothenate. Efficient incorporation into various CoA species was optimized to >99% [(13)C(3)(15)N(1)]-pantothenate after three passages of the murine cells in culture. Charcoal-dextran-stripped fetal bovine serum (FBS) was found to be more efficient for serum supplementation than dialyzed or undialyzed FBS, due to lower contaminating unlabeled pantothenate content. Stable isotope labeled CoA species were extracted and utilized as internal standards for CoA thioester analysis in cell culture models. This methodology of stable isotope labeling by essential nutrients in cell culture (SILEC) can serve as a paradigm for using vitamins and other essential nutrients to generate stable isotope standards that cannot be readily synthesized.

Synthesis and characterization of cis-4-decenoyl-CoA, 3-phenylpropionyl-CoA, and 2,6-dimethylheptanoyl-CoA.

The measurement of acyl-CoA dehydrogenase activities is an essential part of the investigation of patients with suspected defects in fatty acid oxidation. Multiple methods are available for the synthesis of the substrates used for measuring acyl-CoA dehydrogenase activities; however, the yields are low and the products are used without purification. In addition, the reported characterization of acyl-CoAs focuses on the CoA moiety, not on the acyl group. Here we describe the synthesis of three medium-chain acyl-CoAs from mixed anhydrides of the fatty acids using an aqueous-organic solvent mixture optimized to obtain the highest yield. First, cis-4-decenoic acid and 2,6-dimethylheptanoic acid were prepared (3-phenylpropionic acid is commercially available). These were characterized by gas chromatography/mass spectrometry (GC/MS), (1)H nuclear magnetic resonance (NMR), and (13)C NMR. Then cis-4-decenoyl-CoA, 3-phenylpropionyl-CoA, and 2,6-dimethylheptanoyl-CoA were synthesized. These were then purified by ion exchange solid-phase extraction using 2-(2-pyridyl)ethyl-functionalized silica gel, followed by reversed-phase semipreparative high-performance liquid chromatography with ultraviolet detection (HPLC-UV). The purified acyl-CoAs were characterized by analytical HPLC-UV followed by data-dependent tandem mass spectrometry (MS/MS) analysis on the largest responding MS mass (HPLC-UV-MS-MS/MS) and (13)C NMR. The yields of the purified acyl-CoAs were between 75% and 78% based on coenzyme A trilithium salt (CoASH). Acyl-CoA dehydrogenase activities were measured in rat skeletal muscle mitochondria using, as substrates, the synthesized cis-4-decenoyl-CoA, 3-phenylpropionyl-CoA, and 2,6-dimethylheptanoyl-CoA. These results were compared with the results using our standard substrates butyryl-CoA, octanoyl-CoA, and palmitoyl-CoA.

Monitoring enzyme catalysis in the multimeric state: direct observation of Arthrobacter 4-hydroxybenzoyl-coenzyme A thioesterase catalytic complexes using time-resolved electrospray ionization mass spectrometry.

The ability to examine real-time reaction kinetics for multimeric enzymes in their native state may offer unique insights into understanding the catalytic mechanism and its interplay with three-dimensional structure. In this study, we have used a time-resolved electrospray mass spectrometry approach to probe the kinetic mechanism of 4-hydroxybenzoyl-coenzyme A (4-HBA-CoA) thioesterase from Arthrobacter sp. strain SU in the millisecond time domain. Intact tetrameric complexes of 4-HBA-CoA thioesterase with up to four natural substrate (4-HBA-CoA) molecules bound were detected at times as early as 6 ms using an online rapid-mixing device directly coupled to an electrospray ionization time-of-flight mass spectrometer. Species corresponding to the formation of a folded tetramer of the thioesterase at charge states 16+, 17+, 18+, and 19+ around m/z 3800 were observed and assigned as individual tetramers of thioesterase and noncovalent complexes of the tetramers with up to four substrate and/or product molecules. Real-time evaluation of the reaction kinetics was accomplished by monitoring change in peak intensity corresponding to the substrate and product complexes of the tetrameric protein. The mass spectral data suggest that product 4-HBA is released from the active site of the enzyme prior to the release of product CoA following catalytic turnover. This study demonstrates the utility of this technique to provide additional molecular details for an understanding of the individual enzyme states during the thioesterase catalysis and ability to observe real-time interactions between enzyme and substrates and/or products in the millisecond time range.

Acetyl coenzyme A carboxylase: filamentous nature of the animal enzymes.

Acetyl coenzyme A carboxylases purified from several animal tissues exist as enzymatically active polymeric filaments of high molecular weight and have simillar electron microscopic, hydrodynamic, and catalytic properties. These filaments reversibly dissociate into inactive protomers of uniform size. Their re-assembly into catalytically active filaments is promoted by the presence of an allosteric activator.

Silver coordination complex amplified electrochemiluminescence sensor for sensitive detection of coenzyme A and histone acetyltransferase activity.

A kind of coenzyme A (CoA)-silver coordination complex (CoA-Ag) was in-situ developed and verified to accelerate the electron transferring and electrochemical catalysis of H2O2 decomposition to enhance the cathode ECL intensity of CdTe@CdS QDs. Afterward, a convenient label-free signal-on ECL approach was constructed for CoA detection with excellent specificity. In addition, the unique ECL enhancing phenomenon was also proposed to assay the enzymatic activity of histone acetyltransferases (HAT) and screen relevant inhibitors, exhibiting a promising potential in the practical application of biochemical research, disease diagnosis and drug discovery.

An orthogonal purification strategy for isolating crosslinked domains of modular synthases.

Chemo-enzymatic methods for covalently crosslinking carrier proteins with partner enzymes within modular synthases hold promise for elucidating and engineering metabolic pathways. Our efforts to crystallize the ACP-KS complexes of fatty acid synthases have been complicated by difficulties in the purification of the crosslinked complex from the other proteins in the reaction. Here we present a solution that employs an orthogonal purification strategy to achieve the quantity and level of purity necessary for further studies of this complex.

Endogenous coenzyme A glutathione disulfide in human myocardial tissue.

Besides its role as a mechanical pump, the human heart serves as an endocrine organ, where known and as yet unknown hormones are produced. It is very likely that these hormones play an important role in cardiovascular regulation. In this study, a new endogenous vasoactive substance, coenzyme A glutathione disulfide (CoASSG), was isolated and identified in myocardial tissue. Human myocardial tissue was extracted with perchloric acid and fractionated by size exclusion-, displacement-, anion-exchange- and reversed-phase chromatography. In one fraction purified to homogeneity, CoASSG was identified by matrix assisted laser desorption/ionization (MALDI) mass-spectrometry, post-source decay MALDI-mass spectrometry and enzymatic structure analysis. Furthermore, CoASSG was also isolated from human cardiac specific granules. CoASSG has potent vasoconstrictive and proliferative effects. Therefore, CoASSG may affect myocardial function as an endocrine or autocrine substance after being released from myocardial specific granules.

A convenient and rapid method for the complete removal of CoA from butyryl-CoA dehydrogenase.

The commercially available gel, 2-pyridyl disulphide hydroxypropyl ether-Sepharose (thiopropyl-Sepharose 6B), can be used to remove bound ligand completely from butyryl-CoA dehydrogenase (EC 1.399.2) in two simple operations. The resultant enzyme forms normal complexes with acetoacetyl-CoA and CoA persulphide, contains no bound CoA as determined by the enzymatic assay for CoA, and retains full catalytic activity.

The regulation of fatty acid chain elongation in rat liver microsomes: role of fasting and CoASH.

The elongation system for palmitic acid in rat liver microsomes was decreased to 1/3 by fasting, while the elongation of eicosapentaenoic acid was not sensitive to fasting. The rate of eicosapentaenoic acid elongation in the fed state was 50% higher than using palmitic acid as a substrate. The saturated and polyunsaturated fatty acyl-CoA substrates exhibited positive cooperativity on the rate-limiting condensing step in the elongation system, with a Hill constant of approx. 2. An inhibition by CoASH on the total elongation reaction as well as on the condensation step was demonstrated using acyl-CoA substrates, and followed a hyperbolic pattern. The concentrations giving a 50% inhibition (30-70 microM) were in the range found in rat hepatocyte cytosol, indicating that free CoASH has the potential to act as a physiological regulator.

Isolation and characterization of coenzyme A glutathione disulfide as a parathyroid-derived vasoconstrictive factor.

BACKGROUND: Coenzyme A glutathione disulfide (CoA-SSG) was recently isolated from bovine adrenal glands and was shown to be a renal vasoconstrictor. The identification of CoA-SSG in human parathyroid glands and its action on cultured vascular smooth muscle cells (VSMCs) are described here. METHODS AND RESULTS: After purification to homogeneity by several chromatographic steps, CoA-SSG was identified by matrix-assisted laser desorption/ionization mass spectrometry and enzymatic analysis. The dose-dependent growth-stimulating effect of CoA-SSG on VSMCs, measured by the [(3)H]thymidine method, is characterized by a threshold of 10(-)(8) mol/L and a maximum effect of 10 micromol/L, increasing VSMC proliferation 254+/-21% above control. A dose of 10 micromol/L methylmalonyl-CoA and 10 micromol/L CoA increased the rate of proliferation of VSMCs only by 178+/-43% and 50+/-42% above control, respectively. Glutathione has no proliferative effect on VSMCs. The growth-stimulating effect of CoA-SSG (1 micromol/L) was decreased by the antagonists 3,7-dimethyl-1-propargylxanthine (DMPX; 11 micromol/L) (38% compared with CoA-SSG without antagonist) and pyridoxal-phosphate-6-azophenyl-2,4-disulfonic acid (PPADS; 10 micromol/L) (48% compared with CoA-SSG without antagonist; each P:<0. 05 versus control), indicating that the effect is mediated partly via A(2) and partly via P(2)Y(1) and/or P(2)Y(4) receptor. CONCLUSIONS: CoA-SSG may play a regulatory role in VSMC growth as a progression factor and thereby could play an important role in development of hypertension.

Measurement of bile acid CoA esters by high-performance liquid chromatography-electrospray ionisation tandem mass spectrometry (HPLC-ESI-MS/MS).

The novel and rapid assay presented here combines high-performance liquid chromatography and electrospray ionisation tandem mass spectrometry (HPLC-ESI-MS/MS) to directly measure and quantify the CoA esters of 3alpha,7alpha,12alpha-trihydroxy- and 3alpha,7alpha-dihydroxy-5beta-cholestan-26-oic acid (THCA and DHCA). The latter are converted inside peroxisomes to the primary bile acids, cholic and chenodeoxycholic acids, respectively. Prior to MS/MS, esters were separated by reversed-phase HPLC on a C(18) column using an isocratic mobile phase (acetonitrile/water/2-propanol) and subsequently detected by multiple reaction monitoring. For quantification, the CoA ester of deuterium-labelled 3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-oic acid (d(4)-CA) was used as internal standard. To complete an assay took less than 8 min. To verify the validity of the assay, the effect of peroxisomal proteins on the efficacy of extraction of the CoA esters was tested. To this end, variable amounts of the CoA esters were spiked with a fixed amount of either intact peroxisomes or peroxisomal matrix proteins and then extracted using a solid-phase extraction system. The CoA esters could be reproducibly recovered in the range of 0.1-4 micromol l(-1) (linear correlation coefficient R(2) > 0.99), with a detection limit of 0.1 micromol l(-1). In summary, electrospray ionization tandem mass spectrometry combined with HPLC as described here proved to be a rapid and versatile technique for the determination of bile acid CoA esters in a mixture with peroxisomal proteins. This suggests this technique to become a valuable tool in studies dealing with the multi-step biosynthesis of bile acids and its disturbances in disorders like the Zellweger syndrome.