Two biologically active isomers of dihydroouabain isolated from a commercial preparation.

Ouabain is a plant-derived cardiac glycoside that inhibits the catalytic activity of Na(+),K(+)-ATPase (sodium pump; NKA). Dihydroouabain, a derivative of ouabain with a reduced lactone ring, is commonly used as a sodium pump antagonist. It has been assumed that commercially available dihydroouabain is homogeneous. We now report that preparations of dihydroouabain contain two components each with a different potency for inhibition of sodium pump activity. We used reverse-phase HPLC chromatography, UV spectrophotometry, electrospray ionization-mass spectrometry (ESI-MS), nuclear magnetic resonance (NMR) spectroscopy and two independent bioassays to characterize these compounds. The two dihydroouabain fractions (Dho-A and Dho-B) resolved by 3 min chromatographically, had UV absorbance maxima at 196 nm, and comprised 37% and 63% of the stock dihydroouabain, respectively. The molar potency of each component for inhibition of NKA from porcine cerebral cortex differed by 4. 4-fold (Dho-A, IC(50) = 7.13 +/- 0.8 microM; Dho-B, IC(50) = 1.63 +/- 0.12 microM). The relative potencies were 9% and 40% of those of ouabain, respectively. A similar pattern for phosphorylation of NKA was observed. Mass spectrometry (ESI-MS) and fragmentation patterns are consistent with Dho-A and Dho-B being isomers of identical molecular mass (587 Da) and each with six hydroxyl groups, a deoxyhexose sugar moiety and a lactone ring. Furthermore, NMR spectroscopy revealed structural differences between Dho-A and Dho-B by displaying noticeably different chemical shifts at only two groups of proton resonances assigned to H-21 and H-22. The ESI-MS and NMR results confirm the presence of the isomerism at C20 of the lactone ring. Our results demonstrate the existence of two molecular forms of dihydroouabain, each with a different biological potency. These findings underscore the importance of characterizing the purity of dihydroouabain commercial preparations. It also provides possible molecular models for investigating the metabolism of endogenous ouabain-like factors recently reported in mammals.

Characterization of P-S bond hydrolysis in organophosphorothioate pesticides by organophosphorus hydrolase.

The extensive use of organophosphorothioate insecticides in agriculture has resulted in the risk of environmental contamination with a variety of broadly based neurotoxins that inhibit the acetylcholinesterases of many different animal species. Organophosphorus hydrolase (OPH, EC 3.1.8.1) is a broad-spectrum phosphotriesterase that is capable of detoxifying a variety of organophosphorus neurotoxins by hydrolyzing various phosphorus-ester bonds (P-O, P-F, P-CN, and P-S) between the phosphorus center and an electrophilic leaving group. OPH is capable of hydrolyzing the P-X bond of various organophosphorus compounds at quite different catalytic rates: P-O bonds (kcat = 67-5000 s-1), P-F bonds (kcat = 0.01-500 s-1), and P-S bonds (kcat = 0.0067 to 167 s-1). P-S bond cleavage was readily demonstrated and characterized in these studies by quantifying the released free thiol groups using 5,5'-dithio-bis-2-nitrobenzoic acid or by monitoring an upfield shift of approximately 31 ppm by 31P NMR. A decrease in the toxicity of hydrolyzed products was demonstrated by directly quantifying the loss of inhibition of acetylcholinesterase activity. Phosphorothiolate esters, such as demeton-S, provided noncompetitive inhibition for paraoxon (a P-O triester) hydrolysis, suggesting that the binding of these two different classes of substrates was not identical.

Genetically engineered synthesis of precorrin-6x and the complete corrinoid, hydrogenobyrinic acid, an advanced precursor of vitamin B12.

BACKGROUND: Genetically engineered synthesis, in which the gene products, cofactors, and substrates of a complete pathway are combined in vitro in a single flask to give the target, can be a viable alternative to conventional chemical construction of molecules of complex structure and stereochemistry. We chose to attempt to synthesize the metal-free corrinoid hydrogenobyrinic acid, an advanced precursor of vitamin B12. RESULTS: Cloning and overexpression of the genes necessary for the S-adenosyl methionine dependent conversion of 5-aminolevulinic acid (ALA) to precorrin-3 and those required for the synthesis of hydrogenobyrinic acid from precorrin-3 completed the repertoire of the 12 biosynthetic enzymes involved in corrin synthesis. Using these enzymes and the necessary cofactors, the multi-enzyme synthesis of hydrogenobyrinic acid from ALA can be achieved in 20% overall yield in a single reaction vessel, corresponding to an average of at least 90% conversion for each of the 17 steps involved. CONCLUSIONS: By replacing the cell wall with glass, and by mixing the soluble biosynthetic enzymes and necessary cofactors, the major segment of the physiological synthesis of vitamin B12 has been accomplished. Since only those enzymes necessary for the synthesis of hydrogenobyrinic acid from ALA are supplied, none of the intermediates is deflected from the direct pathway. This results in an efficiency which in fact surpasses that of nature.

Biosynthesis of vitamin B12. Discovery of the enzymes for oxidative ring contraction and insertion of the fourth methyl group.

In the vitamin B12 biosynthetic pathway the enzymes responsible for the conversion of precorrin-3 to precorrin-4 have been identified as the gene products of cobG and cobJ from Pseudomonas denitrificans. CobG catalyzes the oxidation of precorrin-3 to precorrin-3x (a hydroxy lactone) whereas CobJ is a SAM-dependent C-17 methyl transferase and is necessary for ring contraction. A mechanism for ring contraction is proposed.

Expression of 9 Salmonella typhimurium enzymes for cobinamide synthesis. Identification of the 11-methyl and 20-methyl transferases of corrin biosynthesis.

Nine of the cbi genes from the 17.5 kb cob operon of Salmonella typhimurium previously shown by genetic studies to be involved in the biosynthesis of cobinamide from precorrin-2, have been subcloned and expressed in Escherichia coli. Seven of the gene products were found in the soluble fraction of cell lysates and have been purified. The gene products corresponding to cbi E, F, H and L were shown by SAM binding and by homology with other SAM-binding proteins to be candidates for the methyltransferases of vitamin B12 biosynthesis. The enzymatic functions of the gene products of cbiL and cbiF are associated with C-methylation at C-20 of precorrin-2 and C-11 of precorrin-3.

Enzymatic synthesis and structure of precorrin-3, a trimethyldipyrrocorphin intermediate in vitamin B12 biosynthesis.

The trimethylated intermediate of vitamin B12 (corrin) biosynthesis, precorrin-3, was produced from various 13C-enriched isotopomers of 5-aminolevulinic acid (ALA), using a multiple-enzyme system containing ALA dehydratase, porphobilinogen deaminase, uro'gen III synthetase, and the S-adenosyl-L-methionine-(SAM)-dependent uro'gen III methyltransferase (M-1) and precorrin-2 methyltransferase (M-2) in the presence of [13C]SAM. Structural analysis of the resulting product, precorrin-3, reveals a close similarity to precorrin-2 but with several subtle differences in the conjugated array of C = C and C = N bonds which reflect the presence of the new C-methyl group at C20 and its influence on the electronic distribution in the dipyrrocorphin chromophore. The implications of this structure for corrin biosynthesis are discussed.

Porcine cytosolic aspartate aminotransferase reconstituted with [4'-13C]pyridoxal phosphate. pH- and ligand-induced changes of the coenzyme observed by 13C NMR spectroscopy.

Apoenzyme samples of aspartate aminotransferase (AspAT) purified from the cytosolic fraction of pig heart were reconstituted with [4'-13C]pyridoxal 5'-phosphate (pyridoxal-P). The 13C NMR spectra of AspAT samples thus generated established the chemical shift of 165.3 ppm for C4' of the coenzyme bound as an internal aldimine with lysine 258 of the enzyme at pH 5. In the absence of ligands the chemical shift of C4' was shown to be pH dependent, shifting 5 ppm upfield to a constant value of 160.2 ppm above pH 8, the resulting pKa of 6.3 in agreement with spectrophotometric titrations. The addition of the competitive inhibitor succinate to the internal aldimine raises the pKa of the imine to 7.8, consistent with the theory of charge neutralization in the active site. In the presence of saturating concentrations of 2-methylaspartic acid the C4' signal of the coenzyme was shown to be invariant with pH and located at 162.7 ppm, midway between the observed chemical shifts of the protonated and unprotonated forms of the internal aldimine. The intermediate chemical shift of the external aldimine complex is thought to reflect the observation of an equilibrium mixture composed of roughly equal populations of the protonated ketoenamine and a dipolar anion species, corresponding to their respective spectral bands at 430 and 360-370 nm. Conversion to the pyridoxamine form was accomplished via reaction of the internal aldimine with L-cysteinesulfinate or by reduction with sodium borohydride, and the resulting C4' chemical shifts were identified by difference spectroscopy. Finally, the line widths of the C4' resonance under the various conditions were measured and qualitatively compared. The results are discussed in terms of the current mechanism and molecular models of the active site of AspAT.

Site-directed mutagenesis and high-resolution NMR spectroscopy of the active site of porphobilinogen deaminase.

The active site of porphobilinogen (PBG)1 deaminase (EC 4.3.1.8) from Escherichia coli has been found to contain an unusual dipyrromethane derived from four molecules of 5-aminolevulinic acid (ALA) covalently linked to Cys-224, one of the two cysteine residues conserved in E. coli and human deaminase. By use of a hemA- strain of E. coli the enzyme was enriched from [5-13C]ALA and examined by 1H-detected multiple quantum coherence spectroscopy, which revealed all of the salient features of a dipyrromethane composed of two PBG units linked head to tail and terminating in a CH2-S bond to a cysteine residue. Site-specific mutagenesis of Cys-99 and Cys-242, respectively, has shown that substitution of Ser for Cys-99 does not affect the enzymatic activity, whereas substitution of Ser for Cys-242 removes essentially all of the catalytic activity as measured by the conversion of the substrate PBG to uro'gen I. The NMR spectrum of the covalent complex of deaminase with the suicide inhibitor 2-bromo-[2,11-13C2]PBG reveals that the aninomethyl terminus of the inhibitor reacts with the enzyme's cofactor at the alpha-free pyrrole. NMR spectroscopy of the ES2 complex confirmed a PBG-derived head-to-tail dipyrromethane attached to the alpha-free pyrrole position of the enzyme. A mechanistic rationale for deaminase is presented.

Identification of a cysteine residue as the binding site for the dipyrromethane cofactor at the active site of Escherichia coli porphobilinogen deaminase.

The dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase was specifically labelled with 13C by growth of the bacteria in the presence of 5-amino[5-13C]levulinic acid. Using 13C-NMR spectroscopy, the structure of the cofactor was confirmed as a dipyrromethane made up of two linked pyrrole rings each derived from porphobilinogen. The chemical shift data indicate that one of the pyrrole rings of the cofactor is covalently linked to the deaminase enzyme through a cysteine residue. Evidence from protein chemistry studies suggest that cysteine-242 is the covalent binding site for the cofactor.