Archaeoglobus fulgidus CbiX^S 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 CbiX^S 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 CbiX^S 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, CbiX^S was observed to catalyze metal insertion for six different combinations of metal and tetrapyrrole. Based upon the turnover numbers of these reactions, CbiX^S 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 CbiX^S 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, CbiX^S is an encouraging starting point for synthetic chelatase design that could yield non-natural metal tetrapyrroles for use in the biotechnology sector.