THE 2ND ACONITASE (ACNB) OF ESCHERICHIA-COLI

The second aconitase (AcnB) of Escherichia coli was partially purified from an acnA::kan(R) mutant lacking AcnA, and the corresponding polyp eptide identified by activity staining and weak cross-reactivity with AcnA antiserum. The acnB gene was located at 2.85 min (131.6 kb) in a region of the chromosome previously assigned to two unidentified ORFs. Aconitase specific activities were amplified up to fivefold by infect ion with lambda acnB phages from the Kohara lambda-E. coli gene librar y, and up to 120-fold (50% of soluble protein) by inducing transforman ts containing a plasmid (pGS783) in which the acnB coding region is ex pressed from a regulated T7 promoter. The AcnB protein was purified to greater than or equal to 98% homogeneity from a genetically enriched source (JRG3171) and shown to be a monomeric protein of M(r) 100000 (S DS-PAGE) and 105000 (gel filtration analysis) compared with M(r) 93 50 0 predicted from the nucleotide sequence, The sequence identity betwee n AcnA and AcnB is only 17% and the domain organization of AcnA and re lated proteins (1-2-3-linker-4) is rearranged in AcnB (4-1-2-3).