Hydrogenobyrinic Acid Research Articles

Two Distinct Thermodynamic Gradients for Cellular Metalation of Vitamin B12.

The acquisition of CoII by the corrin component of vitamin B12 follows one of two distinct pathways, referred to as early or late CoII insertion. The late insertion pathway exploits a CoII metallochaperone (CobW) from the COG0523 family of G3E GTPases, while the early insertion pathway does not. This provides an opportunity to contrast the thermodynamics of metalation in a metallochaperone-requiring and a metallochaperone-independent pathway. In the metallochaperone-independent route, sirohydrochlorin (SHC) associates with the CbiK chelatase to form CoII-SHC. CoII-buffered enzymatic assays indicate that SHC binding enhances the thermodynamic gradient for CoII transfer from the cytosol to CbiK. In the metallochaperone-dependent pathway, hydrogenobyrinic acid a,c-diamide (HBAD) associates with the CobNST chelatase to form CoII-HBAD. Here, CoII-buffered enzymatic assays indicate that CoII transfer from the cytosol to HBAD-CobNST must somehow traverse a highly unfavorable thermodynamic gradient for CoII binding. Notably, there is a favorable gradient for CoII transfer from the cytosol to the MgIIGTP-CobW metallochaperone, but further transfer of CoII from the GTP-bound metallochaperone to the HBAD-CobNST chelatase complex is thermodynamically unfavorable. However, after nucleotide hydrolysis, CoII transfer from the chaperone to the chelatase complex is calculated to become favorable. These data reveal that the CobW metallochaperone can overcome an unfavorable thermodynamic gradient for CoII transfer from the cytosol to the chelatase by coupling this process to GTP hydrolysis.

Optimization of Hydrogenobyrinic Acid Synthesis in a Cell-Free Multienzyme Reaction by Novel S-Adenosyl-methionine Regeneration.

Hydrogenobyrinic acid, a modified tetrapyrrole composed of eight five-carbon compounds, is a key intermediate and central framework of vitamin B12. Synthesis of hydrogenobyrinic acid requires eight S-adenosyl-methionine working as the methyl group donor catalyzed by 12 enzymes including six methyltransferases, causing the great shortage of S-adenosyl-methionine and accumulation of S-adenosyl-homocysteine, which is uneconomic and unsustainable for the cascade reaction. Here, we report a cell-free synthetic system for producing hydrogenobyrinic acid by integrating 12 enzymes using 5-aminolevulininate as a substrate and develop a novel S-adenosyl-methionine regeneration system to steadily supply S-adenosyl-methionine and avoid the accumulated inhibition of S-adenosyl-homocysteine by consuming a cheaper substrate (l-methionine and polyphosphate). By combination of the reaction system optimization and S-adenosyl-methionine regeneration, the titer of hydrogenobyrinic acid was improved from 0.61 to 29.39 mg/L in a 12 h reaction period, representing an increase of 48.18-fold, raising an efficient and rapidly evolutional alternative method to produce high-value-added compounds and intermediate products.

Optimization of hydrogenobyrinic acid biosynthesis in Escherichia coli using multi-level metabolic engineering strategies

BackgroundHydrogenobyrinic acid is a key intermediate of the de-novo aerobic biosynthesis pathway of vitamin B12. The introduction of a heterologous de novo vitamin B12 biosynthesis pathway in Escherichia coli offers an alternative approach for its production. Although E. coli avoids major limitations that currently faced by industrial producers of vitamin B12, such as long growth cycles, the insufficient supply of hydrogenobyrinic acid restricts industrial vitamin B12 production.ResultsBy designing combinatorial ribosomal binding site libraries of the hemABCD genes in vivo, we found that their optimal relative translational initiation rates are 10:1:1:5. The transcriptional coordination of the uroporphyrinogen III biosynthetic module was realized by promoter engineering of the hemABCD operon. Knockdown of competitive heme and siroheme biosynthesis pathways by RBS engineering enhanced the hydrogenobyrinic acid titer to 20.54 and 15.85 mg L−1, respectively. Combined fine-tuning of the heme and siroheme biosynthetic pathways enhanced the hydrogenobyrinic acid titer to 22.57 mg L−1, representing a remarkable increase of 1356.13% compared with the original strain FH215-HBA.ConclusionsThrough multi-level metabolic engineering strategies, we achieved the metabolic balance of the uroporphyrinogen III biosynthesis pathway, eliminated toxicity due to by-product accumulation, and finally achieved a high HBA titer of 22.57 mg L−1 in E. coli. This lays the foundation for high-yield production of vitamin B12 in E. coli and will hopefully accelerate its industrial production.

An enzyme-trap approach allows isolation of intermediates in cobalamin biosynthesis

The biosynthesis of many vitamins and coenzymes has often proved difficult to elucidate due to a combination of low abundance and kinetic lability of the pathway intermediates. Through a serial reconstruction of the cobalamin (vitamin B12) pathway in E. coli, and by His-tagging the terminal enzyme in the reaction sequence, we have observed that many unstable intermediates can be isolated as tightly-bound enzyme-product complexes. Together, these approaches have been used to extract intermediates between precorrin-4 and hydrogenobyrinic acid in their free acid form and permitted the delineation of the overall reaction catalysed by CobL, including the formal elucidation of precorrin-7 as a metabolite. Furthermore, a substrate-carrier protein, CobE, has been identified, which can also be used to stabilize some of the transient metabolic intermediates and enhance their onward transformation. The tight association of pathway intermediates with enzymes provides evidence for a form of metabolite channeling.

The AAA+ motor complex of subunits CobS and CobT of cobaltochelatase visualized by single particle electron microscopy

Cobalamins belong to the tetrapyrrole family of prosthetic groups. The presence of a metal ion is a key feature of these compounds. In the oxygen-dependent (aerobic) cobalamin biosynthetic pathway, cobalt is inserted into a ring-contracted tetrapyrrole called hydrogenobyrinic acid a,c-diamide (HBAD) by a cobaltochelatase that is constituted by three subunits, CobN, CobS and CobT, with molecular masses of 137, 37 and 71kDa, respectively. Based on the similarities with magnesium chelatase, cobaltochelatase has been suggested to belong to the AAA(+) superfamily of proteins. In this paper we present the cloning of the Brucella melitensis cobN, cobS and cobT, the purification of the encoded protein products, and a single-particle reconstruction of the macromolecular assembly formed between CobS and CobT from negatively stained electron microscopy images of the complex. The results show for the first time that subunits CobS and CobT form a chaperone-like complex, characteristic for the AAA(+) class of proteins. The molecules are arranged in a two-tiered ring structure with the six subunits in each ring organized as a trimer of dimers. The similarity between this structure and that of magnesium chelatase, as well as analysis of the amino acid sequences confirms the suggested evolutionary relationship between the two enzymes.

Demonstration That CobG, the Monooxygenase Associated with the Ring Contraction Process of the Aerobic Cobalamin (Vitamin B12) Biosynthetic Pathway, Contains an Fe-S Center and a Mononuclear Non-heme Iron Center

The ring contraction process that occurs during cobalamin (vitamin B(12)) biosynthesis is mediated via the action of two enzymes, CobG and CobJ. The first of these generates a tertiary alcohol at the C-20 position of precorrin-3A by functioning as a monooxygenase, a reaction that also forms a gamma lactone with the acetic acid side chain on ring A. The product, precorrin-3B, is then acted upon by CobJ, which methylates at the C-17 position and promotes ring contraction of the macrocycle by catalyzing a masked pinacol rearrangement. Here, we report the characterization of CobG enzymes from Pseudomonas denitrificans and Brucella melitensis. We show that both contain a [4Fe-4S] center as well as a mononuclear non-heme iron. Although both enzymes are active in vivo, the P. denitrificans enzyme was found to be inactive in vitro. Further analysis of this enzyme revealed that the mononuclear non-heme iron was not reducible, and it was concluded that it is rapidly inactivated once it is released from the bacterial cell. In contrast, the B. melitensis enzyme was found to be fully active in vitro and the mononuclear non-heme iron was reducible by dithionite. The reduced mononuclear non-heme was able to react with the oxygen analogue NO, but only in the presence of the substrate precorrin-3A. The cysteine residues responsible for binding the Fe-S center were identified by site-directed mutagenesis. A mechanism for CobG is presented.

Identification and Characterization of a Novel Vitamin B12 (Cobalamin) Biosynthetic Enzyme (CobZ) from Rhodobacter capsulatus, Containing Flavin, Heme, and Fe-S Cofactors

One of the most intriguing steps during cobalamin (vitamin B12) biosynthesis is the ring contraction process that leads to the extrusion of one of the integral macrocyclic carbon atoms from the tetrapyrrole-derived framework. The aerobic cobalamin pathway requires the action of a monooxygenase called CobG (precorrin-3B synthase), which generates a hydroxylactone intermediate that is subsequently ring-contracted by CobJ. However, in the photosynthetic bacterium Rhodobacter capsulatus, which harbors an aerobic-like pathway, there is no cobG in the main cobalamin biosynthetic operon although it does contain an additional uncharacterized gene called orf663. To demonstrate the involvement of Orf663 in cobalamin synthesis, the first dedicated 10 genes of the B12 pathway (including orf663), encoding enzymes for the transformation of uroporphyrinogen III into hydrogenobyrinic acid (HBA), were sequentially cloned into a plasmid to generate an artificial operon, which, when transformed into Escherichia coli, endowed the host with the ability to make HBA. Deletion of orf663 from this operon prevented HBA synthesis, demonstrating that it was essential for corrin construction. HBA synthesis was restored to this recombinant strain either by returning orf663 or by substituting it with cobG. Recombinant overproduction of Orf663, now renamed CobZ, allowed the characterization of a novel cofactor-rich protein, housing two Fe-S centers, a flavin, and a heme group, which like B12 itself is a modified tetrapyrrole. A mechanism for Orf663 (CobZ) in cobalamin biosynthesis is proposed.

Crystal structure of a predicted precorrin‐8x methylmutase from Thermoplasma acidophilum

The biosynthesis of vitamin B12 involves many enzymatic steps and two evolutionarily divergent pathways. Although the pathways differ in their requirement for molecular oxygen and the timing of cobalt insertion, the precorrin-8x methylmutase genes cobH and cbiC share greater than 30% identity, and are designated as the CobH family.1 These enzymes catalyze the methyl isomerization of metal-free (CobH) and cobalt bound (CbiC) precorrin-8x to hydrogenobyrinic acid (HBA). We have determined the 2.1-A crystal structure of precorrin-8x methylmutase from the facultative anaerobe Thermoplasma acidophilum (Ta0654). Although cobalamin synthesis has not been investigated in T. acidophilum, the structure bears a striking resemblance to CobH from Pseudomonas denifitricans (Pa2905), to which it shares 33% sequence identity. The structure–function relationships for Pa2905 have been characterized, including the role of catalytic residues and crystallographic observation of the product HBA in the active site formed by dimerization.2–4

Crystal Structure of Precorrin-8x Methyl Mutase

Background: The crystal structure of precorrin-8x methyl mutase (CobH), an enzyme of the aerobic pathway to vitamin B 12, provides evidence that the mechanism for methyl migration can plausibly be regarded as an allowed [1,5]-sigmatropic shift of a methyl group from C-11 to C-12 at the C ring of precorrin-8x to afford hydrogenobyrinic acid. Results: The dimeric structure of CobH creates a set of shared active sites that readily discriminate between different tautomers of precorrin-8x and select a discrete tautomer for sigmatropic rearrangement. The active site contains a strictly conserved histidine residue close to the site of methyl migration in ring C of the substrate. Conclusion: Analysis of the structure with bound product suggests that the [1,5]-sigmatropic shift proceeds by protonation of the ring C nitrogen, leading to subsequent methyl migration.

Vitamin B12. Recent discoveries cast new light on an ancient structure

Abstract

Absolute Stereochemistry of Precorrin-3x and Its Relevance to the Dichotomy of Ring Contraction Mechanism in Vitamin B12 Biosynthesis

In Nature, two pathways of vitamin B12 biosynthesis have been discovered. In the aerobe Pseudomonas denitrificans molecular oxygen is utilized in the formation of the novel hydroxy-γ-lactone intermediate precorrin-3x. In the subsequent conversion of precorrin-3x to hydrogenobyrinic acid catalyzed by the Ps. denitrificans Cob enzymes, we demonstrate that no oxygen exchange of the peripheral carboxylic acid functions with the medium takes place. This result is in sharp contrast to the anaerobic pathway in Propionibacterium shermanii, where a unique carboxylate function (ring A acetate) undergoes extensive oxygen exchange. The stereochemistry of precorrin-3x at the C-20 center was determined using NMR and it is proposed that the C-20 hydroxyl is cis to the oxygen terminus of the γ-lactone at C-1, a result that has a distinct bearing on the ring contraction mechanism of corrin biosynthesis in Ps. denitrificans. The implications of these findings for both the aerobic and anaerobic pathways are discussed and tw...

Overexpression in Escherichia coli of 12 Vitamin B 12 Biosynthetic Enzymes

The first 12 enzymes involved in the biosynthesis of vitamin B 12 from its five-carbon precursor, aminolevulinic acid, have been overexpressed in recombinant strains of Escherichia coli. The activity of each enzyme has been demonstrated by the biosynthesis of hydrogenobyrinic acid from aminolevulinic acid.

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

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. 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. 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.

Assay, purification, and characterization of cobaltochelatase, a unique complex enzyme catalyzing cobalt insertion in hydrogenobyrinic acid a,c-diamide during coenzyme B12 biosynthesis in Pseudomonas denitrificans.

Hydrogenobyrinic acid a,c-diamide was shown to be the substrate of cobaltochelatase, an enzyme that catalyzes cobalt insertion in the corrin ring during the biosynthesis of coenzyme B12 in Pseudomonas denitrificans. Cobaltochelatase was demonstrated to be a complex enzyme composed of two different components of M(r) 140,000 and 450,000, which were purified to homogeneity. The 140,000-M(r) component was shown to be coded by cobN, whereas the 450,000-M(r) component was composed of two polypeptides specified by cobS and cobT. Each component was inactive by itself, but cobaltochelatase activity was reconstituted upon mixing CobN and CobST. The reaction was ATP dependent, and the Km values for hydrogenobyrinic acid a,c-diamide, Co2+, and ATP were 0.085 +/- 0.015, 4.2 +/- 0.2, and 220 +/- 36 microM, respectively. Spectroscopic data revealed that the reaction product was cob(II)yrinic acid a,c-diamide, and experiments with a coupled-enzyme incubation system containing both cobaltochelatase and cob(II)yrinic acid a,c-diamide reductase (F. Blanche, L. Maton, L. Debussche, and D. Thibaut, J. Bacteriol. 174:7452-7454, 1992) confirmed this result. This report not only provides the first evidence that hydrogenobyrinic acid and its a,c-diamide derivative are indeed precursors of adenosylcobalamin but also demonstrates that precorrin-6x, precorrin-6y, and precorrin-8x, three established precursors of hydrogenobyrinic acid (D. Thibaut, M. Couder, A. Famechon, L. Debussche, B. Cameron, J. Crouzet, and F. Blanche, J. Bacteriol. 174:1043-1049, 1992), are also on the pathway to cobalamin.

The final step in the biosynthesis of hydrogenobyrinic acid is catalyzed by the cobH gene product with precorrin-8x as the substrate

The final enzymatic reaction in the conversion of precorrin-6x to hydrogenobyrinic acid by cell-free protein preparations from Pseudomonas denitrificans was shown to be inhibited by hydrogenobyrinic acid. Use was made of this property to prepare the last biosynthetic precursor of hydrogenobyrinic acid, named precorrin-8x. Double-labeling experiments, mass spectrometry, and UV-visible light spectroscopy studies established that precorrin-8x was at the oxidation level of a corrin and differed from precorrin-6x by two additional methyl groups (presumably at C-5 and C-15) and decarboxylation of the acetic acid side chain at C-12. Precorrin-8x was not a corrin but had the same mass as hydrogenobyrinic acid, thus showing that this latter compound is synthesized from the former by a rearrangement. The enzyme catalyzing this rearrangement was purified 80-fold to homogeneity from a recombinant strain of P. denitrificans, sequenced at its N terminus, and shown to be encoded by the cobH gene. It was identical to the previously described hydrogenobyrinic acid-binding protein (F. Blanche, D. Thibaut, D. Frechet, M. Vuilhorgne, J. Crouzet, B. Cameron, G. Müller, K. Hlineny, U. Traub-Eberhard, and M. Zboron, Angew. Chem. Int. Ed. Engl. 29:884-886, 1990). This enzyme had a Km of 0.91 +/- 0.04 microM and a Vmax of 230 nmol h-1 mg-1 at pH 7.7 and was competitively inhibited by hydrogenobyrinic acid with a Ki of 0.17 +/- 0.01 microM. It is proposed that the cobH gene product is a mutase which transfers the methyl group from C-11 to C-12.

Precorrin-6x reductase from Pseudomonas denitrificans: purification and characterization of the enzyme and identification of the structural gene

Precorrin-6x reductase, which catalyzes the NADPH-dependent reduction of precorrin-6x to a dihydro derivative named precorrin-6y, was purified 14,300-fold to homogeneity with an 8% yield from extracts of a recombinant strain of Pseudomonas denitrificans. Precorrin-6y was identified by fast atom bombardment-mass spectrometry. It was converted in high yield (90%) to hydrogenobyrinic acid by cell-free protein preparations from P. denitrificans. For the purification and characterization of precorrin-6x reductase, a coupled-enzyme radioenzymatic assay was developed in which precorrin-6y was methylated in situ by the cobL gene product (F. Blanche, A. Famechon, D. Thibaut, L. Debussche, B. Cameron, J. Crouzet, J. Bacteriol. 174:1050-1052, 1992) in the presence of [methyl-3H]S-adenosyl-L-methionine. Molecular weights of precorrin-6x reductase obtained by gel filtration (Mr congruent to 27,000) and by analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr congruent to 31,000) were consistent with the enzyme being a monomer. Km values of 3.6 +/- 0.2 microM for precorrin-6x and 23.5 +/- 3.5 microM for NADPH and a Vmax value of 17,000 U mg-1 were obtained at pH 7.7. The N-terminal sequence (six amino acids) and three internal sequences obtained after tryptic digestion of the enzyme were determined by microsequencing and established that precorrin-6x reductase is encoded by the cobK gene, located on a previously described 8.7-kb EcoRI fragment (J. Crouzet, B. Cameron, L. Cauchois, S. Rigault, M.-C. Rouyez, F. Blanche, D. Thibaut, and L. Debussche, J. Bacteriol. 172:5980-5990, 1990). However, the coding sequence was shown to be on the strand complementary to the one previously proposed as the coding strand.

Biosynthesis of vitamin B12: structure of the ester of a new biosynthetic intermediate, precorrin-6y

13 C Labelling and NMR experiments establish the structure of precorrin-6y octamethyl ester, the corresponding octa-acid being a further new intermediate on the biosynthetic pathway to hydrogenobyrinic acid 1.

Biosynthesis of vitamin B12: stereochemistry of transfer of a hydride equivalent from NADPH by precorrin-6x reductase

Deuterium labelling with NMR analysis demonstrates that the enzyme precorrin-6x reductase transfers HR from C-4 of the pyridine residue of NADPH as the first step in the sequence leading from precorrin-6x to hydrogenobyrinic acid.

Biosynthesis of vitamin B12: the site of reduction of precorrin-6x

It is proved by deuterium labelling and NMR spectroscopy that a hydride equivalent from NADPH is transferred to C-19 of precorrin-6x as this intermediate is converted enzymically into hydrogenobyrinic acid.

Biosynthesis of vitamin B12: isolation of precorrin-6x, a metal-free precursor of the corrin macrocycle retaining five S-adenosylmethionine-derived peripheral methyl groups.

delta-Aminolevulinic acid and trimethylisobacteriochlorin are converted by cell-free protein preparations from Pseudomonas denitrificans into a metal-free pigment, precorrin-6x. This pigment, which accumulates when the cell-free system lacks NADPH, can be enzymically converted in high yield (greater than 50%) into hydrogenobyrinic acid by the complete enzyme preparation. Double-labeling experiments establish that precorrin-6x carries five C-methyl groups, which appear at C-1, C-2, C-7, C-12 alpha, and C-17 of the hydrogenobyrinic acid formed enzymically from precorrin-6x. This precursor of the corrin macrocycle is at the dehydrocorrin level of oxidation, has undergone ring contraction and extrusion of C-20, but still carries the acetic acid side chain at C-12. It is demonstrated that the conversion of precorrin-6x into hydrogenobyrinic acid specifically requires an NADPH-dependent reduction step.