Archaeoglobus fulgidus CbiXS is a promiscuous chelatase with a broad substrate scope.

Organisms biosynthesize square planar metal tetrapyrrole cofactors containing Mg, Fe, Co, and Ni using chelatase enzymes, which selectively insert the correct metal into a specific organic ligand. Archaeoglobus fulgidus CbiXS has been proposed to be an "ancestral" chelatase enzyme; in this work we have evaluated its substrate scope to ascertain whether this enzyme is a promising starting point for synthetic chelatase design. Size-exclusion chromatography revealed that CbiXS is a tetramer in solution regardless of the metalation state of the enzyme, in contrast to the dimeric structure of the nickel chelatase CfbA. Reaction conditions that minimize enzyme-free metal insertion into tetrapyrroles were determined, which revealed that many of the in vitro activity assays reported in the literature for chelatase enzymes may be plagued by non-enzymatic metal insertion. Under these conditions that minimize uncatalyzed metal insertion, CbiXS was observed to catalyze metal insertion for six different combinations of metal and tetrapyrrole. Based upon the turnover numbers of these reactions, CbiXS is most effective at catalyzing metal insertion for reactants similar to its native substrates: cobalt and sirohydrochlorin. Kinetic analysis for reactions with varying substrate concentrations revealed that CbiXS may be substrate-inhibited for non-native substrates. In addition, the kinetic data identifies metal-ligand bond formation as the rate-limiting step of the enzyme. Due to its broad substrate scope, CbiXS is an encouraging starting point for synthetic chelatase design that could yield non-natural metal tetrapyrroles for use in the biotechnology sector.