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PubMed1996

Achieving natural product synthesis and diversity via catalytic networking ex vivo.

Recent studies on ex vivo synthesis of natural products reveal that even complex multistep pathways can be successfully reconstructed. Genetic engineering of such reconstituted pathways has already been used to generate 'unnatural' natural products related to the original compound. In the future, it may be possible to use these approaches to make natural products that are currently inaccessible to conventional synthesis.

Carbohydrates↗
PubMed1995

Mechanistic studies of the methyltransferase from Clostridium thermoaceticum: origin of the pH dependence of the methyl group transfer from methyltetrahydrofolate to the corrinoid/iron-sulfur protein.

A methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) from Clostridium thermoaceticum catalyzes the transfer of the N5 methyl group from (6S)-methyltetrahydrofolate (CH3-H4folate) to the cobalt center of a corrinoid/iron-sulfur protein (C/Fe-SP). The methylcobamide product is the first in a series of enzyme-bound organometallic intermediates in the acetyl-CoA pathway of anaerobic CO2 fixation. The mechanisms of the forward and reverse reactions with CH3-H4folate and either the C/Fe-SP or vitamin B12 as substrates were studied by steady-state and pre-steady-state kinetics. This ability to effectively utilize free cobalamin as well as the C/Fe-SP in the transmethylation appears to explain why [14C]methylcobyric acid was found as a product of labeling C. thermoaceticum cells with 14CO2 [Ljungdahl, L. G., Irion, E., & Wood, H. G. (1965) Biochemistry 4, 2771-2780]. Stopped-flow experiments indicate that the Co(I)-C/Fe-SP performs a direct SN2 displacement of the methyl group of CH3-H4folate to form H4folate and methyl-Co(III). The pre-steady-state rate constants in the forward and reverse reactions increased as the pH was lowered (pKa approximately 5.5). Similar pH profiles were obtained by steady-state kinetics. The kcat/Km values for the C/Fe-SP and CH3-H4folate in the forward direction and for the methylated C/Fe-SP and H4folate in the reverse direction increased as the pH was lowered (pKa approximately 5.3). A different pH profile was obtained with free cobalamin as the substrate; the kcat/Km for CH3-H4folate and cobalamin (forward reaction) increased (pKa approximately 7.0) and the kcat/Km for H4folate and methylcobalamin (reverse reaction) decreased (pKa approximately 5.3) as the pH was lowered.(ABSTRACT TRUNCATED AT 250 WORDS)

Clostridium↗
PubMed1986

Biosynthesis of porphyrins and corrins. 1. 1H and 13C NMR spectra of (hydroxymethyl)bilane and uroporphyrinogens I and III.

High-field NMR spectroscopic methods have been applied to study the reactions catalyzed by porphobilinogen (PBG) deaminase and uroporphyrinogen III (uro'gen III) cosynthase, which are the enzymes responsible for the formation of the porphyrin macrocycle. The action of these enzymes in the conversion of PBG, [2,11-13C]PBG, and [3,5-13C]PBG to uro'gens I and III has been followed by 1H and 13C NMR, and assignments are presented. The principal intermediate that accumulated was the correspondingly labeled (hydroxymethyl)bilane (HMB), the assignments for which are also presented.

Ammonia-Lyases↗
PubMed1986

Biosynthesis of porphyrins and corrins. 2. Isolation, purification, and NMR investigations of the porphobilinogen-deaminase covalent complex.

The procedures for the generation of enzyme-substrate complexes from labeled porphobilinogens [(2,11-13C]PBG and [2,6,11-3H]PBG) with deaminase and the methods employed for their purification are described. Use of 13C NMR failed to detect the substrate bound to the enzyme, suggesting that the line width must be inordinately large. The complex was found to disproportionate with time when stored at 25 degrees C. However, enzyme-bound uroporphyrinogen I (uro'gen I) was detected, both in the intact protein and in the oligopeptides from tryptic digestion and peptide mapping. The first detection of an enzyme-substrate complex by 3H NMR is described for [3H]PBG and deaminase. The line widths of the observed resonances were found to be extremely large and dependent upon temperature, giving chemical shifts that suggest the involvement of a sulfhydryl group as the nucleophilic enzyme group that binds the substrate. The catalytic competence of this complex was also demonstrated by displacing bound [3H]PBG with unlabeled PBG. During the resultant formation of [3H]uro'gen I, a transient low-intensity signal was detected that has been tentatively assigned to the highly reactive azafulvene species, proposed in several mechanistic schemes for porphyrin biosynthesis.

Ammonia-Lyases↗
PubMed1996

A conformational change in the methyltransferase from Clostridium thermoaceticum facilitates the methyl transfer from (6S)-methyltetrahydrofolate to the corrinoid/iron-sulfur protein in the Acetyl-CoA pathway.

The methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) from Clostridium thermoaceticum catalyzes the methylation of a corrinoid/iron-sulfur protein (C/Fe-SP) by the N5 methyl group of (6S)-methyltetrahydrofolate (CH3-H4folate). This is an important reaction in the reductive acetyl-CoA pathway. The forward and reverse reactions of MeTr have a pH dependence that appears to reflect protonation of a group on the protein [Zhao, S., Roberts, D. L., & Ragsdale, S. W. (1995) Biochemistry 34, 15075-15083]. In the work reported here, fluorescence and rapid reaction kinetics were used to demonstrate that this protonation elicits a rate-limiting conformational change. As the pH was lowered, the emission maximum for intrinsic tryptophan fluorescence underwent a red shift (pK(a) = 5.4) and the emission intensity increased (pK(a) = 5.1). The extrinsic fluorescence probe, 4,4'-bis-1-phenylamino-8-napthalenesulfonate (bis-ANS) was used to report on the conformational change. The bis-ANS fluorescence was strongly enhanced upon binding MeTr. As the pH was decreased, the fluorescence was further enhanced and the emission maximum underwent a 14 nm blue shift (pK(a) = 5.0). By stopped-flow fluorescence studies, it was shown that these fluorescence changes occur at rates similar to the k(cat) for the MeTr reaction and thus reflect catalytically competent events. The combined results indicate that CH3-H4folate binds to a hydrophobic region in MeTr that includes a tryptophan residue(s). MeTr undergoes a pH-dependent conformational change that exposes this region to solvent and facilitates substrate binding.

Acetyl Coenzyme A↗
PubMed1998

Definition of the redox states of cobalt-precorrinoids: investigation of the substrate and redox specificity of CbiL from Salmonella typhimurium.

The enzyme CbiL from the facultative anaerobe Salmonella typhimurium exhibits a high degree of homology to CobI from the aerobe Pseudomonas denitrificans (29% identity; 51% conservation obtained by a Blastp search of the ncbi database). As CobI catalyzes the third methylation in the aerobic pathway to vitamin B12 it is proposed that CbiL catalyzes the analogous step in the anaerobic pathway. Potential metallo and metal-free substrates were characterized and their redox states defined by a combination of physicochemical techniques (MALDI-MS, NMR, UV/vis, IR, and EPR) and then used to investigate the function of CbiL. CbiL exhibited an absolute requirement for the presence of a metal ion (Co(II), Ni(II), or Zn(II)) within the tetrapyrrole substrate. CbiL had no preference for the redox state of its cobalt tetrapyrrole substrate, methylating both the reduced form, Co(II) 2, 7-dimethyl-dipyrrocorphin (Co(II)-precorrin-2), and the oxidized form, Co(III) 2,7-dimethyl-isobacterioclorin (Co(III)-factor-II). In contrast CbiL had a marked preference for the oxidized Ni(II) and Zn(II)-2,7-dimethyl-isobacteriochlorin (Ni(II) and Zn(II)-factor-II). Removal of the metal ion from a product of CbiL (Zn(II)-factor-III) allowed characterization by 13C NMR, identifying the tetrapyrrole as 2,7,20-trimethyl-isobacteriochlorin (factor-3), indicating that CbiL methylates at C20, the same site as that methylated by CobI. Competition experiments, utilizing isotopic labeling to distinguish otherwise identical mass substrates and products, revealed that oxidized Co(III) or Ni(II)-factor-II were equally good substrates, whereas Co(II)-precorrin-2 was much preferred over Ni(II)-precorrin-2. Excess Ni(II)-precorrin-2 did not decrease CbiL methylation of Co(II)-precorrin-2, implying that CbiL has a low affinity for Ni(II)-precorrin-2. These results are interpreted on the basis of tetrapyrrole ruffling occurring on the optimization of the metallo-N bond distances. The greater flexibility of the reduced precorrin-2 ring system allows greater deformation on accommodating the bound metal ion, the distortions imposed by bound Ni(II) or Zn(II) ions being larger than Co(II). The resulting distortions imposed on the precorrin ring could then decrease catalysis by causing a departure from the optimal substrate conformation required for CbiL. On oxidation of the Ni(II) or Zn(II)-precorrin-2, the increased stiffness of the ring could then constrain the metallo-factor-II conformation toward that of the usual substrate, allowing greater methylation by CbiL. In contrast to its counterpart CobI in the aerobic pathway of B12 biosynthesis, which methylates the metal-free precorrin-2, these studies show CbiL to be the first methylase unique to the anaerobic pathway, methylating a metallo-precorrin-2 substrate. Implications of CbiL specificity for the mechanism of the anaerobic B12 pathway are discussed.

Bacterial Proteins↗
PubMed1999

Mechanism of transfer of the methyl group from (6S)-methyltetrahydrofolate to the corrinoid/iron-sulfur protein catalyzed by the methyltransferase from Clostridium thermoaceticum: a key step in the Wood-Ljungdahl pathway of acetyl-CoA synthesis.

The methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) from Clostridium thermoaceticum catalyzes transfer of the N5-methyl group from (6S)-methyltetrahydrofolate (CH3-H4folate) to the cobalt center of a corrinoid/iron-sulfur protein (CFeSP), forming methylcob(III)amide and H4folate. This reaction initiates the unusual biological organometallic reaction sequence that constitutes the Wood-Ljungdahl or reductive acetyl-CoA pathway. The present paper describes the use of steady-state, product inhibition, single-turnover, and kinetic simulation experiments to elucidate the mechanism of the MeTr-catalyzed reaction. These experiments complement those presented in the companion paper in which binding and protonation of CH3-H4folate are studied by spectroscopic methods [Seravalli, J., Shoemaker, R. K., Sudbeck, M. J., and Ragsdale, S. W. (1999) Biochemistry 38, 5736-5745]. Our results indicate that a pH-dependent conformational change is required for methyl transfer in the forward and reverse directions; however, this step is not rate-limiting. CH3-H4folate and the CFeSP [in the cob(I)amide state] bind randomly and independently to form a ternary complex. Kinetic simulation studies indicate that CH3-H4folate binds to MeTr in the unprotonated form and then undergoes rapid protonation. This protonation enhances the electrophilicity of the methyl group, in agreement with a 10-fold increase in the pKa at N5 of CH3-H4folate. Next, the Co(I)-CFeSP attacks the methyl group in a rate-limiting SN2 reaction to form methylcob(III)amide. Finally, the products randomly dissociate. The following steady-state constants were obtained: kcat = 14.7 +/- 1.7 s-1, Km of the CFeSP = 12 +/- 4 microM, and Km of (6S)-CH3-H4folate = 2.0 +/- 0.3 microM. We assigned the rate constants for the elementary reaction steps by performing steady-state and pre-steady-state kinetic studies at different pH values and by kinetic simulations.

Acetyl Coenzyme A↗
PubMed2003

Axial solvent coordination in "base-fff" cob(II)alamin and related co(II)-corrinates revealed by 2D-EPR.

Detailed information on the structure of cobalt(II) corrinates is of interest in the context of studies on the coenzyme B(12) catalyzed enzymatic reactions, where cob(II)alamin has been identified as a reaction intermediate. Cob(II)ester (heptamethyl cobyrinate perchlorate) is found to be soluble in both polar and nonpolar solvents and is therefore very suitable to study solvent effects on Co(II) corrinates. In the literature, Co(II) corrinates in solution are often addressed as four-coordinated Co(II) corrins. However, using a combination of continuous-wave (CW) and pulse electron paramagnetic resonance (EPR) and pulse ENDOR (electron nuclear double resonance) at different microwave frequencies we clearly prove axial ligation for Cob(II)ester and the base-off form of cob(II)alamin (B(12r)) in different solvents. This goal is achieved by the analysis of the g values, and the hyperfine couplings of cobalt, some corrin nitrogens and hydrogens, and solvent protons. These parameters are shown to be very sensitive to changes in the solvent ligation. Density functional computations (DFT) facilitate largely the interpretation of the EPR data. In the CW-EPR spectrum of Cob(II)ester in methanol, a second component appears below 100 K. Different cooling experiments suggest that this observation is related to the phase transition of methanol from the alpha-phase to the glassy state. A detailed analysis of the EPR parameters indicates that this transition induces a change from a five-coordinated (above 100 K) to a six-coordinated (below 100 K) Co(II) corrin. In a CH(3)OH:H(2)O mixture the phase-transition properties alter and only the five-coordinated form is detected for Cob(II)ester and for base-off B(12r) at all temperatures. Our study thus shows that the characteristics of the solvent can have a large influence on the structure of Co(II) corrinates and that comparison with the protein-embedded cofactor requires some caution. Finally, the spectral similarities between Cob(II)ester and base-off B(12r) prove the analogies in their electronic structure.

Cobalt↗
PubMed2003

Discovering nature's diverse pathways to vitamin B12: a 35-year odyssey.

The chronology of the discoveries along the pathway of vitamin B(12) biosynthesis is reviewed from a personal perspective, including discussion of the most recent finding that two pathways to B(12) exist-one aerobic and one anaerobic-which differ mainly in the ring contraction mechanisms that convert porphyrin to corrin.

Aerobiosis↗
PubMed2005

Photophysical properties of a series of free-base corroles.

Four free-base corroles with electron-donating or electron-withdrawing groups on the para or 2 through 6-positons of the meso phenyl rings were prepared via either Paolesse or Gross conditions and investigated for their absorption and emission properties. The triaryl corroles 5,10,15-triphenylcorrole, 5,10,15-tris(pentafluorophenyl)corrole, 5,10,15-tris(p-nitrophenyl)corrole, and 5,10,15-tris(p-methoxyphenyl)corrole were examined. Absorption, steady-state, and time-resolved fluorescence measurements were performed on all compounds in both nonpolar (dichloromethane) and polar (dimethylacetamide) solvents. The experimental evidence points to hydrogen bonding with an internal N-H group as the most likely factor in the solvent-dependent photophysical behavior of these corroles, that is also highly dependent upon substitution.

Corrinoids↗
PubMed1996

Confirmation of the Mukerjee term in an extended Corrin-Harkins relation using an anionic amphiphilic drug.

The amphiphilic behavior of the S-enantiomer of 3(S)-[(2-carboxyethyl)sulfanyl]-3-[2-(8-phenyloctyl)phenyl] propionic acid disodium salt (C26H32O4S.2Na) has been investigated as a function of the ionic strength, mu (buffer and simple electrolyte). The linearity predicted by the Corrin-Harkins relation (J. Am. Chem. Soc. 1947, 69, 683-688) is observed at low buffer concentrations, and it is verified that the nature of the buffer is of less importance than the concentration of the counterion. At high counterion concentrations, the salting-out effect predicted by Mukerjee in 1965 (J. Phys. Chem. 1965, 69, 4038-4040) and 1967 (Adv. Coll. Interf. Sci. 1967, 1, 241-275) manifests itself, and a more complete equation resulting from attaching a salting-out term to the Corrin-Harkins relation, as used recently by Mukerjee and Chan (ACS Abstracts of Papers 1993, 206, COLL 164; Ph.D. Thesis, University of Wisconsin, 1993), gives a better representation of the experimental data. It is shown that the parameter values obtained from this equation are reasonable and predict self-association in the form of micelles rather than as small aggregates.

Anions↗