Stamford N P, Crouzet J, Cameron B, Alanine A I, Pitt A R, Yeliseev A A, Battersby A R
University Chemical Laboratory, University of Cambridge, U.K.
Biochem J. 1996 Jan 1;313 ( Pt 1)(Pt 1):335-42. doi: 10.1042/bj3130335.
The Bacillus subtilis genes hemB, hemC and hemD, encoding respectively the enzymes porphobilinogen synthase, hydroxymethylbilane synthase and uroporphyrinogen III synthase, have been expressed in Escherichia coli using a single plasmid construct. An enzyme preparation from this source converts 5-aminolaevulinic acid (ALA) preparatively and in high yield into uroporphyrinogen III. The Pseudomonas denitrificans genes cobA and cobI, encoding respectively the enzymes S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT) and S-adenosyl-L-methionine:precorrin-2 methyltransferase (SP2MT), were also expressed in E. coli. When SUMT was combined with the coupled-enzyme system that produces uroporphyrinogen III, precorrin-2 was synthesized from ALA, and when SP2MT was also added the product from the coupling of five enzymes was precorrin-3A. Both of these products are precursors of vitamin B12, and they can be used directly for biosynthetic experiments or isolated as their didehydro octamethyl esters in > 40% overall yield. The enzyme system which produces precorrin-3A is sufficiently stable to allow long incubations on a large scale, affording substantial quantities (15-20 mg) of product.
枯草芽孢杆菌的hemB、hemC和hemD基因分别编码胆色素原合酶、羟甲基胆色素合酶和尿卟啉原III合酶，利用单一质粒构建体在大肠杆菌中进行了表达。来自该来源的酶制剂可将5-氨基乙酰丙酸（ALA）大量且高产率地制备转化为尿卟啉原III。反硝化假单胞菌的cobA和cobI基因分别编码S-腺苷-L-甲硫氨酸：尿卟啉原III甲基转移酶（SUMT）和S-腺苷-L-甲硫氨酸：前咕啉-2甲基转移酶（SP2MT），它们也在大肠杆菌中进行了表达。当SUMT与产生尿卟啉原III的偶联酶系统结合时，可从ALA合成前咕啉-2，而当加入SP2MT时，五种酶偶联的产物为前咕啉-3A。这两种产物都是维生素B12的前体，它们可直接用于生物合成实验，或以其双脱氢八甲基酯的形式分离得到，总产率超过40%。产生前咕啉-3A的酶系统足够稳定，能够进行大规模长时间孵育，从而获得大量（15 - 20毫克）产物。
