Monomeric class II chelatase with four histidine residues at the active site, designated Mch4 chelatase, shows evolutionary trails from ancestral to descendant-types

Abstract

Class II chelatases catalyze the insertion of divalent metal ions into tetrapyrroles during the biosynthesis of metal-tetrapyrroles, and are regarded as standard models for investigating chelation mechanisms. The catalytic core of class II chelatases exhibits either a homodimer- or a monomer-type architecture with similar overall folds and different catalytic His residues: four His residues in the homodimer type and one or two His residues in the monomer type. These structural features of the two types of chelatases have led to the hypothesis that the monomer-type is a “descendant” evolved from the homodimer-type “ancestor”. However, there has been no report on naturally-occurring “evolutionary intermediate”-type of chelatases to support this hypothesis. Here, we show the discovery and characterization of such “evolutionary intermediate”-type chelatases. Because this type of chelatases was classified into monomeric chelatases with four histidine residues at the active site, we named these chelatases Mch4. Gene complement analysis showed Mch4s could play a role in Fe²⁺ insertion into sirohydrochlorin. Notably, Mch4s did not utilize coproporphyrin III or protoporphyrin IX in vivo assay. In addition, in vitro functional analysis of Mch4s demonstrated that they could utilize Ni²⁺, Co²⁺ as well as Fe²⁺ insertion into sirohydrochlorin. These findings suggest that nickel chelatase activity with sirohydrochlorin was maintained in the hypothetical class II chelatase evolution from homodimeric ancestor-type, to monomeric evolutionary-intermediate-type Mch4, followed by changes in the metal and tetrapyrrole preferences of some descendant chelatases through further evolution, such as further substitution of His at their active sites.