Summary auto-generated
This study identifies and characterizes AcnB, the second aconitase enzyme of Escherichia coli. The researchers purified AcnB from an acnA-null mutant and located the acnB gene at 2.85 minutes on the chromosome, revealing it derives from two adjacent open reading frames. AcnB is a monomeric protein with a molecular weight of approximately 100,000 Da, containing a [4Fe-4S] iron-sulfur cluster essential for catalytic activity in the citric acid cycle. The enzyme shows only 17% sequence identity to the primary aconitase (AcnA) and exhibits an unusual domain rearrangement compared to related proteins. Using phage-based amplification and plasmid expression systems, researchers achieved up to 120-fold enrichment of aconitase activity, enabling purification to 98% homogeneity. The study demonstrates that AcnB can be reactivated by ferrous ions under reducing conditions, suggesting iron-dependent regulation similar to other aconitases. The findings reveal the existence of functionally distinct aconitases in E. coli with different structural organizations despite catalyzing the same biochemical reaction.
Key findings
- AcnB is the second aconitase of E. coli, a monomeric protein of ~100 kDa with a [4Fe-4S] cluster essential for catalysis
- The acnB gene is located at 2.85 minutes on the chromosome and derives from two adjacent open reading frames with an unusual 4-1-2-3 domain arrangement versus the typical 1-2-3-linker-4 organization
- AcnB shares only 17% sequence identity with AcnA despite catalyzing the same citrate-isocitrate isomerization reaction in the citric acid cycle
- Aconitase activity can be amplified up to 120-fold using plasmid-based T7 expression systems, enabling purification to near-homogeneity
- AcnB activity is reversibly inactivated during purification but can be reactivated by ferrous ions and reducing conditions, indicating iron-dependent regulation
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Abstract
The second aconitase (AcnB) of Escherichia coli was partially purified from an acnA::kanR mutant lacking AcnA, and the corresponding polypeptide identified by activity staining and weak cross-reactivity with AcnA antiserum. The acnB gene was located at 2 center dot 85 min (131 center dot 6 kb) in a region of the chromosome previously assigned to two unidentified ORFs. Aconitase specific activities were amplified up to fivefold by infection with lambdaacnB phages from the Kohara lambda-E. coli gene library, and up to 120-fold (50% of soluble protein) by inducing transformants containing a plasmid (pGS783) in which the acnB coding region is expressed from a regulated T7 promoter. The AcnB protein was purified to > or = 98% homogeneity from a genetically enriched source (JRG3171) and shown to be a monomeric protein of Mr 100 000 (SDS-PAGE) and 105 000 (gel filtration analysis) compared with Mr 93 500 predicted from the nucleotide sequence. The sequence identity between AcnA and AcnB is only 17% and the domain organization of AcnA and related proteins (1-2-3-linker-4) is rearranged in AcnB (4-1-2-3).