Aerobic synthesis of vitamin B12: ring contraction and cobalt chelation

2005 ◽  
Vol 33 (4) ◽  
pp. 815-819 ◽  
Author(s):  
D. Heldt ◽  
A.D. Lawrence ◽  
M. Lindenmeyer ◽  
E. Deery ◽  
P. Heathcote ◽  
...  
Keyword(s):  
Vitamin B12 ◽  
Metal Ion ◽  
Biosynthetic Pathway ◽  
Rhodobacter Capsulatus ◽  
Chlorophyll Synthesis ◽  
Subsequent Reduction ◽  
Ring Contraction ◽  
Contraction Process ◽  
Cobalamin Biosynthesis ◽  
Haem Iron

The aerobic biosynthetic pathway for vitamin B12 (cobalamin) biosynthesis is reviewed. Particular attention is focused on the ring contraction process, whereby an integral carbon atom of the tetrapyrrole-derived macrocycle is removed. Previous work had established that this chemically demanding step is facilitated by the action of a mono-oxygenase called CobG, which generates a hydroxy lactone intermediate. This mono-oxygenase contains both a non-haem iron and an Fe-S centre, but little information is known about its mechanism. Recent work has established that in bacteria such as Rhodobacter capsulatus, CobG is substituted by an isofunctional protein called CobZ. This protein has been shown to contain flavin, haem and Fe-S centres. A mechanism is proposed to explain the function of CobZ. Another interesting aspect of the aerobic cobalamin biosynthetic pathway is cobalt insertion, which displays some similarity to the process of magnesium chelation in chlorophyll synthesis. The genetic requirements of cobalt chelation and the subsequent reduction of the metal ion are discussed.

scholarly journals Characterization of the Enzyme CbiH60Involved in Anaerobic Ring Contraction of the Cobalamin (Vitamin B12) Biosynthetic Pathway

2012 ◽  
Vol 288 (1) ◽  
pp. 297-305 ◽  
Author(s):  
Simon J. Moore ◽  
Rebekka Biedendieck ◽  
Andrew D. Lawrence ◽  
Evelyne Deery ◽  
Mark J. Howard ◽  
...  
Keyword(s):  
Vitamin B12 ◽  
Biosynthetic Pathway ◽  
Ring Contraction

scholarly journals ATPase activity associated with the magnesium chelatase H-subunit of the chlorophyll biosynthetic pathway is an artefact

2006 ◽  
Vol 400 (3) ◽  
pp. 477-484 ◽  
Author(s):  
Nick Sirijovski ◽  
Ulf Olsson ◽  
Joakim Lundqvist ◽  
Salam Al-Karadaghi ◽  
Robert D. Willows ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Molecular Mass ◽  
Mass Fraction ◽  
Biosynthetic Pathway ◽  
Rhodobacter Capsulatus ◽  
Gel Filtration ◽  
Protoporphyrin Ix ◽  
High Molecular Mass ◽  
Magnesium Chelatase ◽  
Ring Complex

Magnesium chelatase inserts Mg2+ into protoporphyrin IX and is the first unique enzyme of the chlorophyll biosynthetic pathway. It is a heterotrimeric enzyme, composed of I- (40 kDa), D- (70 kDa) and H- (140 kDa) subunits. The I- and D-proteins belong to the family of AAA+ (ATPases associated with various cellular activities), but only I-subunit hydrolyses ATP to ADP. The D-subunits provide a platform for the assembly of the I-subunits, which results in a two-tiered hexameric ring complex. However, the D-subunits are unstable in the chloroplast unless ATPase active I-subunits are present. The H-subunit binds protoporphyrin and is suggested to be the catalytic subunit. Previous studies have indicated that the H-subunit also has ATPase activity, which is in accordance with an earlier suggested two-stage mechanism of the reaction. In the present study, we demonstrate that gel filtration chromatography of affinity-purified Rhodobacter capsulatus H-subunit produced in Escherichia coli generates a high- and a low-molecular-mass fraction. Both fractions were dominated by the H-subunit, but the ATPase activity was only found in the high-molecular-mass fraction and magnesium chelatase activity was only associated with the low-molecular-mass fraction. We demonstrated that light converted monomeric low-molecular-mass H-subunit into high-molecular-mass aggregates. We conclude that ATP utilization by magnesium chelatase is solely connected to the I-subunit and suggest that a contaminating E. coli protein, which binds to aggregates of the H-subunit, caused the previously reported ATPase activity of the H-subunit.

Biosynthesis of vitamin B12: ring contraction is preceded by incorporation of molecular oxygen into precorrin-3

1993 ◽  
Vol 115 (24) ◽  
pp. 11610-11611 ◽  
Author(s):  
Jonathan B. Spencer ◽  
Neal J. Stolowich ◽  
Charles A. Roessner ◽  
Changhee Min ◽  
A. Ian Scott
Keyword(s):  
Vitamin B12 ◽  
Molecular Oxygen ◽  
Ring Contraction

scholarly journals Identification and functional analysis of enzymes required for precorrin-2 dehydrogenation and metal ion insertion in the biosynthesis of sirohaem and cobalamin in Bacillus megaterium

2003 ◽  
Vol 370 (2) ◽  
pp. 505-516 ◽  
Author(s):  
Evelyne RAUX ◽  
Helen K. LEECH ◽  
Richard BECK ◽  
Heidi L. SCHUBERT ◽  
Patricio J. SANTANDER ◽  
...  
Keyword(s):  
Bacillus Megaterium ◽  
Metal Ion ◽  
Enzyme Assay ◽  
Sequence Alignments ◽  
Rich Region ◽  
In Vitro Synthesis ◽  
Cobalamin Biosynthesis ◽  
High Degree ◽  
Degree Of Similarity

In Bacillus megaterium, the hemAXBCDL genes were isolated and were found to be highly similar to the genes from Bacillus subtilis that are required for the conversion of glutamyl-tRNA into uroporphyrinogen III. Overproduction and purification of HemC (porphobilinogen deaminase) and -D (uroporphyrinogen III synthase) allowed these enzymes to be used for the in vitro synthesis of uroporphyrinogen III from porphobilinogen. A second smaller cluster of three genes (termed sirABC) was also isolated and found to encode the enzymes that catalyse the transformation of uroporphyrinogen III into sirohaem on the basis of their ability to complement a defined Escherichia coli (cysG) mutant. The functions of SirC and -B were investigated by direct enzyme assay, where SirC was found to act as a precorrin-2 dehydrogenase, generating sirohydrochlorin, and SirB was found to act as a ferrochelatase responsible for the final step in sirohaem synthesis. CbiX, a protein found encoded within the main B. megaterium cobalamin biosynthetic operon, shares a high degree of similarity with SirB and acts as the cobaltochelatase associated with cobalamin biosynthesis by inserting cobalt into sirohydrochlorin. CbiX contains an unusual histidine-rich region in the C-terminal portion of the protein, which was not found to be essential in the chelation process. Sequence alignments suggest that SirB and CbiX share a similar active site to the cobaltochelatase, CbiK, from Salmonella enterica.

scholarly journals Identification and Characterization of an Archaeon-Specific Riboflavin Kinase

2008 ◽  
Vol 190 (7) ◽  
pp. 2615-2618 ◽  
Author(s):  
Zahra Mashhadi ◽  
Hong Zhang ◽  
Huimin Xu ◽  
Robert H. White
Keyword(s):  
Escherichia Coli ◽  
Metal Ion ◽  
Biosynthetic Pathway ◽  
Recombinant Expression ◽  
Cell Extract ◽  
Flavin Mononucleotide ◽  
E Coli ◽  
Gene Recombinant ◽  
Identification And Characterization

ABSTRACT The riboflavin kinase in Methanocaldococcus jannaschii has been identified as the product of the MJ0056 gene. Recombinant expression of the MJ0056 gene in Escherichia coli led to a large increase in the amount of flavin mononucleotide (FMN) in the E. coli cell extract. The unexpected features of the purified recombinant enzyme were its use of CTP as the phosphoryl donor and the absence of a requirement for added metal ion to catalyze the formation of FMN. Identification of this riboflavin kinase fills another gap in the archaeal flavin biosynthetic pathway. Some divalent metals were found to be potent inhibitors of the reaction. The enzyme represents a unique CTP-dependent family of kinases.

Revealing the promoting effect of betaine on vitamin B12 biosynthetic pathway of Pseudomonas denitrificans by using a proteomics analysis

2021 ◽  
Vol 22 ◽  
Author(s):  
Kun-tai Li ◽  
Yong Yang ◽  
Xin Cheng
Keyword(s):  
Vitamin B12 ◽  
Biosynthetic Pathway ◽  
Aminolevulinic Acid ◽  
Fermentation Medium ◽  
Promoting Effect ◽  
Pseudomonas Denitrificans ◽  
Methyl Group ◽  
Integral Effect ◽  
Comparative Metabolomics ◽  
Porphobilinogen Synthase

Background: Our previous comparative metabolomics research revealed that betaine (N,N,N-trimethylglycine, a typically essential methyl-group donor for vitamin B12 biosynthesis) had a powerful promoting effect on the generation of vitamin B12 precursors and intermediates in vitamin B12-producing Pseudomonas denitrificans. However, the integral effect of betaine on the vitamin B12 biosynthetic pathway is still unclear. Objective: Considering the vitamin B12 biosynthetic pathway of P. denitrificans as a whole, this work aimed to reveal the biological function of betaine on the vitamin B12 biosynthetic pathway in P. denitrificans, which would sharpen and expand the understanding of betaine as the methyl-group donor for vitamin B12 biosynthesis. Materials and Methods: By using a proteomics method based on the iTRAQ technique, the present study compared and analyzed the differential expression of proteins involved in vitamin B12 biosynthetic pathway under 10 g/L betaine addition to P. denitrificans fermentation medium. Results: The results showed that betaine could significantly up-regulate the expression of proteins related to the vitamin B12 biosynthetic pathway, which was mainly reflected in the following three aspects: 1) the δ-aminolevulinic acid (ALA) synthase and porphobilinogen synthase that were responsible for the formation of the committed precursors for tetrapyrrole-derived macrocycle in vitamin B12 molecule; 2) the C-methylation-related enzymes (such as precorrin-4 C(11)-methyltransferase, Precorrin-2 C(20)-methyltransferase, Precorrin-8X methylmutase, and Precorrin-6Y C5,15-methyltransferase) and methionine synthase that were crucial to the C-methylation reactions for vitamin B12 biosynthesis; 3) the late-stage key enzymes (Cobaltochelatase, and Cob(I)yrinic acid a,c-diamide adenosyltransferase) that were related to cobalt chelation of vitamin B12 molecule. Conclusions: The present study clearly demonstrated that betaine could significantly promote the expression of the integral enzymes involved in the vitamin B12 biosynthetic pathway of P. denitrificans, thus promoting vitamin B12 biosynthesis.

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