Spectroscopic characterization of active-site variants of the PduO-type ATP:corrinoid adenosyltransferase from Lactobacillus reuteri: insights into the mechanism of four-coordinate Co(II)corrinoid formation

The PduO-type adenosine 5'-triphosphate (ATP):corrinoid adenosyltransferase from Lactobacillus reuteri (LrPduO) catalyzes the transfer of the adenosyl-group of ATP to Co(1+)cobalamin (Cbl) and Co(1+)cobinamide (Cbi) substrates to synthesize adenosylcobalamin (AdoCbl) and adenosylcobinamide (AdoCbi(+)), respectively. Previous studies revealed that to overcome the thermodynamically challenging Co(2+) → Co(1+) reduction, the enzyme drastically weakens the axial ligand-Co(2+) bond so as to generate effectively four-coordinate (4c) Co(2+)corrinoid species. To explore how LrPduO generates these unusual 4c species, we have used magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectroscopic techniques. The effects of active-site amino acid substitutions on the relative yield of formation of 4c Co(2+)corrinoid species were examined by performing eight single-amino acid substitutions at seven residues that are involved in ATP-binding, an intersubunit salt bridge, and the hydrophobic region surrounding the bound corrin ring. A quantitative analysis of our MCD and EPR spectra indicates that the entire hydrophobic pocket below the corrin ring, and not just residue F112, is critical for the removal of the axial ligand from the cobalt center of the Co(2+)corrinoids. Our data also show that a higher level of coordination among several LrPduO amino acid residues is required to exclude the dimethylbenzimidazole moiety of Co(II)Cbl from the active site than to remove the water molecule from Co(II)Cbi(+). Thus, the hydrophilic interactions around and above the corrin ring are more critical to form 4c Co(II)Cbl than 4c Co(II)Cbi(+). Finally, when ATP analogues were used as cosubstrate, only "unactivated" five-coordinate (5c) Co(II)Cbl was observed, disclosing an unexpectedly large role of the ATP-induced active-site conformational changes with respect to the formation of 4c Co(II)Cbl. Collectively, our results indicate that the level of control exerted by LrPduO over the timing for the formation of the 4c Co(2+)corrinoid intermediates is even more exquisite than previously anticipated.     

Reflections on the discovery of nature's pathways to vitamin B12

The chronology of the discoveries along the pathway of vitamin B12 biosynthesis is reviewed from a personal perspective, including discussion of the most recent finding that two pathways to B12 exist--one aerobic and one anaerobic--which differ mainly in the ring contraction mechanisms which convert porphyrin to corrin.     

Biosynthesis of the 3,4-dihydroxybenzoate moieties of petrobactin by Bacillus anthracis

The biosynthesis of the 3,4-dihydroxybenzoate moieties of the siderophore petrobactin, produced by B. anthracis str. Sterne, was probed by isotopic feeding experiments in iron-deficient media with a mixture of unlabeled and D-[(13)C6]glucose at a ratio of 5:1 (w/w). After isolation of the labeled siderophore, analysis of the isotopomers was conducted via one-dimensional (1)H and (13)C NMR spectroscopy, as well as (13)C-(13)C DQFCOSY spectroscopy. Isotopic enrichment and (13)C-(13)C coupling constants in the aromatic ring of the isolated siderophore suggested the predominant route for the construction of the carbon backbone of 3,4-DHB (1) involved phosphoenol pyruvate and erythrose-4-phosphate as ultimate precursors. This observation is consistent with that expected if the shikimate pathway is involved in the biosynthesis of these moieties. Enrichment attributable to phosphoenol pyruvate precursors was observed at C1 and C6 of the aromatic ring, as well as into the carboxylate group, while scrambling of the label into C2 was not. This pattern suggests 1 was biosynthesized from early intermediates of the shikimate pathway and not through later shikimate intermediates or aromatic amino acid precursors.     

Role of the pyridine nitrogen in pyridoxal 5'-phosphate catalysis: activity of three classes of PLP enzymes reconstituted with deazapyridoxal 5'-phosphate

Pyridoxal 5'-phosphate (PLP; vitamin B(6))-catalyzed reactions have been well studied, both on enzymes and in solution, due to the variety of important reactions this cofactor catalyzes in nitrogen metabolism. Three functional groups are central to PLP catalysis: the C4' aldehyde, the O3' phenol, and the N1 pyridine nitrogen. In the literature, the pyridine nitrogen has traditionally been assumed to be protonated in enzyme active sites, with the protonated pyridine ring providing resonance stabilization of carbanionic intermediates. This assumption is certainly correct for some PLP enzymes, but the structures of other active sites are incompatible with protonation of N1, and, consequently, these enzymes are expected to use PLP in the N1-unprotonated form. For example, aspartate aminotransferase protonates the pyridine nitrogen for catalysis of transamination, while both alanine racemase and O-acetylserine sulfhydrylase are expected to maintain N1 in the unprotonated, formally neutral state for catalysis of racemization and β-elimination. Herein, kinetic results for these three enzymes reconstituted with 1-deazapyridoxal 5'-phosphate, an isosteric analogue of PLP lacking the pyridine nitrogen, are compared to those for the PLP enzyme forms. They demonstrate that the pyridine nitrogen is vital to the 1,3-prototropic shift central to transamination, but not to reactions catalyzed by alanine racemase or O-acetylserine sulfhydrylase. Not all PLP enzymes require the electrophilicity of a protonated pyridine ring to enable formation of carbanionic intermediates. It is proposed that modulation of cofactor electrophilicity plays a central role in controlling reaction specificity in PLP enzymes.     

2'-hydroxypyridoxol, a biosynthetic precursor of vitamins B(6) and B(1) in yeast

In separate experiments cultures of the yeast Saccharomyces cerevisiae ATTC 7752 were grown in the presence of [5',5'-2H2]- or of [2',2',5',5'-2H4]-3-hydroxy-2,4,5-tri(hydroxymethyl)pyridine (i.e., 2'-hydroxypyridoxol). The 2H NMR spectra of the samples of pyridoxamine dihydrochloride and of thiamin chloride hydrochloride that were isolated from the two experiments showed the presence of deuterium at the corresponding sites. Entry of deuterium from the specifically 2H-labeled samples of 2'-hydroxypyridoxol into the predicted sites of pyridoxamine and of the pyrimidine unit of thiamin provides the first unequivocal evidence that, in yeast, 2'-hydroxypyridoxol is an intermediate on the route from a C5-sugar into vitamin B6, and adds to the evidence that pyridoxol serves as a precursor of the pyrimidine unit of thiamin, supplying the C5N unit, C-2',2,N-1,C-6,5,5' as an intact unit.     

New Developments in the Field of Vitamin B12: Reactions of the Cobalt Atom in Corrins and in Vitamin B12 Model Compounds

Studies with vitamin B₁₂ model compounds such as the cobaloximes provide a basis for the understanding of the mode of action of corrinoid coenzymes in enzymatic reactions. They also widen our knowledge of the properties and reactions of organocobalt compounds. The present article outlines the most important nonenzymatic reactions of cobalt in vitamin B₁₂ and in model compounds of the cobaloxime type.     

Biosynthetic study of amphidinolide B

The biosynthetic origins of amphidinoide B (1) were investigated on the basis of 13C-NMR data of 13C-enriched samples obtained by feeding experiments with [1-(13)C], [2-(13)C], and [1,2-(13)C2] sodium acetates in cultures of a dinoflagellate Amphidinium sp. These incorporation patterns suggested that 1 was generated from three successive polyketide chains, an isolated C1 unit from C-2 of acetates, six branched C1 units from C-2 of acetates, and an "m-m" and an "m-m-m" unit derived only from C-2 of acetates. The labeling patterns of amphidinolide B (1) were different from those of amphidinolide H (2), a 26-membered macrolide closely related to 1.     

Trichilin B, a novel limonoid with highly rearranged ring system from Trichilia connaroides

Trichilin B (2), a limonoid having an unprecedented highly rearranged ring system, along with biosynthetically correlated trichilin A (1), was isolated from Trichilia connaroides. The structures were elucidated by spectroscopic analysis. Compound 2 features a unique 9,17-oxygen bridge, while two unprecedented δ-lactone rings fused to rings A and B at C-5, C-9 and ring C at C-8, C-14. Their cytotoxic activities were also evaluated.     

锥形硼烷B5H10X的结构和成键性质

采用从头算分子轨道法对锥型硼烷Ｂ５Ｈ１０Ｘ（Ｘ＝Ｂｅ＾－,Ｂ,Ｃ＾＋,ＢｅＨ,ＣＨ＾２＋,Ｂ＾２－,Ｃ＾－,Ｎ,Ｏ＾＋,ＢＨ＾－,ＣＨ,ＮＨ＾＋和ＯＨ＾２＋）进行了研究,结果表明,端氢和桥氢原子与帽基原子相对位置的变化是由帽基原子和环原子轨道的弥散程度及环的尺寸效应共同决定的,端氢原子对桥氢原子的空间位置有着制约作用。     

Biosynthesis of vitamin B12: the multi-enzyme synthesis of precorrin-4 and factor IV

Background: In order to study the biosynthesis of vitamin B₁₂, it is necessary to produce various intermediates along the biosynthetic pathway by enzymic methods. Recently, information on the organisation of the biosynthetic pathway has permitted the selection of the set of enzymes needed to biosynthesise any specific identified intermediate. The aim of the present work was to use recombinant enzymes in reconstituted multi-enzyme systems to biosynthesise particular intermediates.Results: The products of the cobG and cobJ genes from Pseudomonas denitrificans were expressed heterologously in Escherichia coli to afford good levels of activity of the corresponding enzymes, CobG and CobJ. Aerobic incubation of precorrin-3A with the CobG enzyme alone yielded precorrin-3B. When CobJ and S-adenosyl-l-methionine were included in the incubation, the product was precorrin-4. Both precorrin-3B and precorrin-4 are known precursors of vitamin B₁₂ and their availability has allowed new mechanistic studies of enzymic transformations.Conclusions: Our results show that the expression of the CobG and CobJ enzymes has been successful, thus facilitating the biosynthesis of two precursors of vitamin B₁₂. This lays the foundation for the structure determination of CobG and CobJ as well as future enzymic experiments focusing on later steps of vitamin B₁₂ biosynthesis.     

Homocoenzyme B12 and bishomocoenzyme B12: covalent structural mimics for homolyzed, enzyme-bound coenzyme B12

Efficient electrochemical syntheses of "homocoenzyme B(12)" (2, Co(beta)-(5'-deoxy-5'-adenosyl-methyl)-cob(III)alamin) and "bishomocoenzyme B(12)" (3, Co(beta)-[2-(5'-deoxy-5'-adenosyl)-ethyl]-cob(III)alamin) are reported here. These syntheses have provided crystalline samples of 2 and 3 in 94 and 77 % yield, respectively. In addition, in-depth investigations of the structures of 2 and 3 in solution were carried out and a high-resolution crystal structure of 2 was obtained. The two homologues of coenzyme B(12) (2 and 3) are suggested to function as covalent structural mimics of the hypothetical enzyme-bound "activated" (that is, "stretched" or even homolytically cleaved) states of the B(12) cofactor. From crude molecular models, the crucial distances from the corrin-bound cobalt center to the C5' atom of the (homo)adenosine moieties in 2 and 3 were estimated to be about 3.0 and 4.4 A, respectively. These values are roughly the same as those found in the two "activated" forms of coenzyme B(12) in the crystal structure of glutamate mutase. Indeed, in the crystal structure of 2, the cobalt center was observed to be at a distance of 2.99 A from the C5' atom of the homoadenosine moiety and the latter was found to be present in the unusual syn conformation. In solution, the organometallic moieties of 2 and 3 were shown to be rather flexible and to be considerably more dynamic than the equivalent group in coenzyme B(12). The homoadenosine moiety of 2 was indicated to occur in both the syn and the anti conformations.     

A molecular mechanics force field for the cobalt corrinoids

A force field for the cobalt (III) corrinoids (derivatives of vitamin B₁₂) for use with a modified version of the molecular mechanics program 2(87) has been developed empirically around 19 cobalt corrinoid crystal structures. Bond lengths, bond angles and torsional angles are reproduced with r.m.s. differences of 0.01 Å, 2.4 °, and 4.2 °, respectively, within the standard deviation of the mean of these parameters found in the solid state. The axial ligand occupying the lower coordination site in the cobalamins, 5,6-dimethylbenzimidazole, is shown to have very limited rotational freedom and is constrained by the downward-pointing b and d propionamide side chains of the corrin ring. Strain-energy profiles for rotation of the side chains of the corrin ring show the existence of several local energy minima and this explains the observed variability in the orientations of these side chains in the solid state. The known change in conformation which occurs in the C ring when the e side chain is epimerized from the lower to the upper face of the corrin ring in cyano-13-epicobalamin is correctly predicted, provided the starting conformation of the C ring is unbiased. A study of cyano-8-epicobalamin indicates that an analogous conformational change does not occur in the B ring and the epimerized d side chain assumes an equatorial orientation relative to the corrin ring. Parameters for the Co---C bond in alkylcobalamins were developed and the structure of methyl- and adenosylcobalamin are accurately reproduced. An examination of the strain energy consequences of rotation of the adenosyl ligand about the Co---C bond identifies a number of low-energy conformations at least two of which, in which adenosyl lies over the “southern” and “eastern” portions of the corrin ring, respectively, have been previously deduced from NMR observations. Coordinated neopentyl in neopentylcobalamin is much more hindered to rotation about the Co---C bond and the lowest conformation finds two γ(C) atoms straddling the upwardly projecting C46 methyl group of the corrin.     

Chukrasones A and B: Potential Kv1.2 Potassium Channel Blockers with New Skeletons from Chukrasia tabularis

Two new limonoids, chukrasone A (1) incorporating a highly rearranged A/B ring system and chukrasone B (2) possessing the first 16,19-dinor limonoid backbone with an extended C3 unit at C-15, were isolated from Chukrasia tabularis. Their structures were characterized on the basis of detailed spectroscopic analysis. Compounds 1 and 2 exhibited potential inhibition of the delayed rectifier (I(K)) K(+) current.     

Studies on the biosynthesis of corrinoids and porphyrinoids. I. The labeling of oxygen of vitamin B12

Recently the amide-oxygen has been suggested to participate in the formation of the corrin ring of vitamin B12. To confirm this hypothesis, 17O-labeled aminolevulinic acid (ALA) was prepared and administered to Propionibacterium shermanii. The isolated vitamin B12 showed only broad 17O signals in the oxygen-17 nuclear magnetic resonance (17O-NMR) spectrum. However, distinct isotope-shifted peaks were observed in the 13C-NMR spectrum of vitamin B12 isolated after incorporation of [1-13C:1,4-18O2]ALA. Of these shifted peaks, one peak (C27) showed very low intensity. This indicates that dilution of 18O occurred at the acetyl chain of the A ring of vitamin B12. This result supports the assumption that the lactone formation of the A ring promotes the ring contraction, as proposed by Eschenmoser.     

Biosynthesis of the beta-amino acid moiety of the enediyne antitumor antibiotic C-1027 featuring beta-amino acyl-S-carrier protein intermediates

The enediyne antitumor antibiotic C-1027 chromoprotein is produced by Streptomyces globisporus. The biosynthesis of the (S)-3-chloro-4,5-dihydroxy-beta-phenylalanine moiety (boxed) of the C-1027 chromophore (1) from l-tyrosine (3) and its incorporation into 1 are catalyzed by six enzymes: SgcC, SgcC1, SgcC2, SgcC3, SgcC4, ShcC5. In vivo and in vitro characterization of these enzymes delineated this pathway, unveiling a novel strategy for beta-amino acid modification featuring beta-amino acyl-S-carrier protein intermediates. These findings shed new light into beta-amino acid biosynthesis and present a new opportunity to engineer the C-1027 biosynthetic machinery for the production of novel analogues as exemplified by 20-deschloro-C-1027 (4), 20-deschro-22-deshydroxy-C-1027 (5), and 22-deshydroxy-C-1027 (6).     

Studies on the biosynthesis of corrinoids and porphyrinoids. III. The origin of amide nitrogen of vitamin B12

To clarify the origin of amide-nitrogen of vitamin B12, [1-13C]aminolevulinic acid (ALA) and L-[amide-15N]glutamine were administered to P. shermanii. The 13C-nuclear magnetic resonance spectrum of the vitamin B12 subsequently isolated showed distinct 13C-15N coupling and isotope shift at six amide carbons. However, the C-57 amide carbon showed neither coupling, nor shift. Thus, it was concluded that the nitrogens of 6 amides of the side chain were derived from glutamine and the C-57 amide nitrogen was from threonine.     

Organic Free Radical-Promoted Isomerization of alpha- and beta-Alkylcobinamides(1)

Anaerobic reaction of alpha- or beta-alkylcobinamides (alpha- or beta-RCbi(+)'s) with the corresponding alkyl free radical, R(*) (where R = CH(3), CH(3)CH(2), or CH(3)CH(2)OCH(2)CH(2)), generated by the Fenton reaction using Fe(2+) and an alkyl hydroperoxide, RC(CH(3))(2)OOH, causes isomerization and leads to mixtures of alpha- and beta-RCbi(+)'s. The reaction does not occur, however, under aerobic conditions or under anaerobic conditions in the presence of an excess of the free radical scavenger H-Tempo. In addition, alpha-CH(3)CH(2)Cbi(+) reacts with 50 molar equiv of tert-butyl hydroperoxide and Fe(2+) to give a mixture of alkylcobinamides that contains 6% alpha-CH(3)Cbi(+) and 94% beta-CH(3)Cbi(+), showing that multiple transalkylations occur. A Co(II)-induced isomerization and the S(H)2 mechanism are ruled out on the basis of the known reactivity of RCbi(+) and product analysis. A mechanism is proposed which involves a direct oxidative free radical displacement by an R(*) to the metal of RCbi(+) via a dialkylcobalt(IV) corrinoid species. Since the reaction leads to equilibration of the two diastereomers under mild conditions, it can be used to study the equilibria between diastereomeric RCbi(+)'s. Thus, the equilibrium for the diastereomeric ethyl-13-epicobinamides, in which the e propionamide side chain of the corrin ring has been epimerized from the alpha to the beta face of the corrinoid, lies significantly more toward the alpha diastereomer than that for the normal ethylcobinamides. This represents the most direct experimental evidence obtained to date that the corrin ring side chains control the relative steric accessibility of the two faces of the cobalt corrinoids.     

Biosynthesis of vitamin B1 in yeast. Derivation of the pyrimidine unit from pyridoxine and histidine. Intermediacy of urocanic acid

Incorporation studies with 13C-, 15N-, and 2H-labeled substrates, followed by NMR analysis, show that the pyrimidine unit of thiamin (Vitamin B1) originates from a C5N fragment, derived from C-2',2,N,C-6,5,5' of pyridoxol (Vitamin B6) and an N-C-N fragment derived from L-histidine. Urocanic acid serves as an intermediate on the route of the N-C-N fragment of histidine into the thiamin pyrimidine.     

Identifying the minimal enzymes required for anhydrotetracycline biosynthesis

The cyclohexenone ring A of tetracyclines exhibits unique structural features not observed among other aromatic polyketides. These substitutions include the C2 primary amide, C4 dimethylamine, and the C12a tertiary alcohol. Here we report the identification and reconstitution of the minimum set of enzymes required for the biosynthesis of anhydrotetracycline (ATC, 5), the first intermediate in the tetracycline biosynthetic pathway that contains the fully functionalized ring A. Using a combination of in vivo and in vitro approaches, we confirmed OxyL, OxyQ, and OxyT to be the only enzymes required to convert 6-methylpretetramid 1 into 5. OxyL is a NADPH-dependent dioxygenase that introduces two oxygen atoms into 1 to yield the unstable intermediate 4-keto-ATC 2. The aminotransferase OxyQ catalyzes the reductive amination of C4-keto of 2, yielding 4-amino-ATC 3. Furthermore, the N, N-dimethyltransferase OxyT catalyzes the formation of 5 from 3 in a (S)-adenosylmethionine (SAM)-dependent manner. Finally, a "non-natural" anhydrotetracycline derivative was generated, demonstrating that our heterologous host/vector pair can be a useful platform toward the engineered biosynthesis of tetracycline analogues.     

Highly regioselective anaerobic photocyclization of 3-styrylpyridines.

The photochemical behavior of the cis isomers of the three isomeric styrylpyridines and two (aminostyryl)pyridines has been investigated under aerobic and anaerobic conditions. Both 3-styrylpyridine and its 3'-amino derivative undergo highly regioselective formation of 2-azaphenanthrene products under anaerobic conditions. In the presence of oxygen, mixtures of 4- and 2-azaphenanthrene products are obtained. The formation of 2-azaphenanthrenes in the absence of oxygen is attributed to conversion of the 4a,4b-dihydroazaphenanthrene primary photoproduct to a 1,4-dihydropyridine intermediate by means of a formal 1,7-hydrogen shift. This intermediate is moderately stable in the absence of oxygen and has been characterized by comparison of its (1)H NMR and electronic absorption spectra with calculated spectra. This intermediate is converted to the 2-azaphenanthrene in both the absence and presence of oxygen. The regioselectivity of photocyclization of 3-substituted stilbenes and related diarylethylenes is suggested to depend on the relative rate constants for ring opening and sigmatropic rearrangements of the dihydrophenanthrene intermediates as well as their rates of reaction with oxygen or other oxidants.