A story of chelatase evolution -: Identification and characterization of a small 13-15-kda "ancestral" cobaltochelatase (CbiXs) in the archaea

The cobaltochelatase required for the synthesis of vitamin B-12 ( cobalamin) in the archaeal kingdom has been identified as CbiX through similarity searching with the CbiX from Bacillus megaterium. However, the CbiX proteins in the archaea are much shorter than the CbiX proteins found in eubacteria, typically containing less than half the number of amino acids in their primary structure. For this reason the shorter CbiX proteins have been termed CbiX(S) and the longer versions CbiX(L). The CbiX(S) proteins from Methanosarcina barkeri and Methanobacter thermoautotrophicum were overproduced in Escherichia coli as recombinant proteins and characterized. Through complementation studies of a defined chelatase- deficient strain of E. coli and by direct in vitro assays the function of CbiX(S) as a sirohydrochlorin cobaltochelatase has been demonstrated. On the basis of sequence alignments and conserved active site residues we suggest that CbiX(S) may represent a primordial chelatase, giving rise to larger chelatases such as CbiX(L), SirB, CbiK, and HemH through gene duplication and subsequent variation and selection. A classification scheme for chelatases is proposed.