Stamford N P, Duggan S, Li Y, Alanine A I, Crouzet J, Battersby A R
University Chemical Laboratory, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK.
Chem Biol. 1997 Jun;4(6):445-51. doi: 10.1016/s1074-5521(97)90196-4.
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.
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.
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.
为了研究维生素B12的生物合成，有必要通过酶法生产生物合成途径中的各种中间体。最近，关于生物合成途径组织的信息使得能够选择生物合成任何特定已鉴定中间体所需的一组酶。本研究的目的是在重组多酶系统中使用重组酶来生物合成特定的中间体。
反硝化假单胞菌的cobG和cobJ基因产物在大肠杆菌中异源表达，相应的酶CobG和CobJ具有良好的活性水平。仅将预咕啉-3A与CobG酶进行需氧孵育可产生预咕啉-3B。当孵育中加入CobJ和S-腺苷-L-甲硫氨酸时，产物为预咕啉-4。预咕啉3B和预咕啉-4都是已知的维生素B12前体，它们的可得性使得能够对酶促转化进行新的机理研究。
我们的结果表明，CobG和CobJ酶的表达是成功的，从而促进了维生素B12的两种前体的生物合成。这为CobG和CobJ的结构测定以及未来专注于维生素B12生物合成后续步骤的酶学实验奠定了基础。
