Vitamin B₁₂: How the Problem of Its Biosynthesis Was Solved

Abstract

Vitamin B,, is an essential vitamin for human health, and lack of it leads to pernicious anemia. This biological activity has attracted intense interest for some time; in addition, the complex architecture of the B,, molecule has fascinated chemists and biochemists since its discovery as the first natural organocobalt complex and the establishment of its structure by X-ray analysis. The organic ligand surrounding the cobalt displays many stereogenic centers along its periphery carrying reactive functional groups. This complexity led vitamin B,, to be rightly regarded as an extreme challenge to the synthetic chemist. Yet microorganisms achieve this synthesis in vivo with complete control of regio-and stereochemistry. How do they do it? This review tells the full remarkable story. Success in unraveling this biosynthetic puzzle resulted from a collaborative effort by biologists and chemists using the full range of methods available from their disciplines-from genetics at one end of the spectrum to synthesis and NMR spectroscopy at the other. This work can act as a guide for future research on the biosynthesis of yet more complex natural substances.

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

“…The enzymes required to convert 5-aminolevulinic acid to uroporphyrinogen III are provided by the host, and the large plasmid was transformed into an E. coli strain harboring another plasmid bearing the cobA and cobI genes necessary to convert uroporphyrinogen III to precorrin-3A to provide sufficient amounts of this intermediate and to facilitate high flux through the pathway (10). The resulting strain was found to produce comparatively large quantities of HBA, the first intermediate on the pathway to cobalamin that is stable and can be easily isolated (17). HBA was isolated both as a free acid and in its esterified form and its structure confirmed on the basis of its UV-visible, mass, and NMR spectra (Fig.…”

Identification, Characterization, and Structure/Function Analysis of a Corrin Reductase Involved in Adenosylcobalamin Biosynthesis

“…This enzyme of the cobalamin biosynthetic pathway reduced to the Co(I) state all of the Co(II)-corrinoids isolated from this microorganism, indicating a broad substrate specificity. Despite the N-terminal sequencing, the authors were unable to identify the gene encoding this enzyme (7).…”

Vitamin B 12 - Structure, Biosynthesis , Functions, and Methods of Determination

“…Ta skomplikowana synteza, obejmująca ponad 70 przemian chemicznych, jest jednak zbyt droga i trudna techniczne, aby mogła być wykorzystana na skalę przemysłową [22,49,95]. Obecnie kobalamina produkowana jest jedynie na drodze mikrobiologicznej przy użyciu wyselekcjonowanych i/lub genetycznie zmodyfikowanych drobnoustrojów [5,6,62].…”

“…Badania molekularne dotyczące zwiększenia efektywności biosyntezy witaminy B 12 przez drobnoustroje doprowadziły do wyizolowania szeregu enzymów odpowiedzialnych za tworzenie się tego związku [62]. Szlaki metaboliczne na których syntetyzowana jest witamina B 12 zostały scharakteryzowane w przypadku bakterii Pseudomonas denitrificans [6], Salmonella typhimurium [76] oraz Propionibacterium freudenreichii [73,79]. Geny odpowiedzialne za biosyntezę witaminy B 12 lub jej prekursorów zostały wyizolowane także dla rodzaju Rhodobacter [57,63,64].…”

“…W przeciwień-stwie do bakterii propionowych, proces biosyntezy B 12 przez Pseudomonas denitrificans odbywa się całkowicie w warunkach tlenowych. W wyniku połączenia zabiegów mutagenizacji z jednoczesnym zastosowaniem narzędzi inżynierii genetycznej, grupa naukowców z Rhône-Poulenc-Rorer (RPR) stworzyła wysoce efektywny szczep Pseudomonas denitrificans, za pomocą którego możliwa jest produkcja witaminy B 12 na poziomie 300 mg/l [6]. W europejskim patencie 0516647 B1 Blanche i wsp.…”