Nickel‐chelatase activity of SirB variants mimicking the His arrangement in the naturally occurring nickel‐chelatase CfbA

Abstract

Metal–tetrapyrrole cofactors are involved in multiple cellular functions, and chelatases are key enzymes for the biosynthesis of these cofactors. CfbA is an ancestral, homodimeric‐type class II chelatase which is able to use not only Ni2+ as a physiological metal substrate, but also Co2+ as a nonphysiological substrate with higher activity than for Ni2+. The Ni/Co‐chelatase function found in CfbA is also observed in SirB, a descendant, monomeric‐type class II chelatase. This is despite the distinct active site structure of CfbA and SirB; specifically, CfbA shows a unique four His residue arrangement, unlike other monomeric class II chelatases such as SirB. Herein, we studied the Ni‐chelatase activity of SirB variants R134H, L200H, and R134H/L200H, the latter of which mimics the His alignment of CfbA. Our results showed that the SirB R134H variant exhibited the highest Ni‐chelatase activity among the SirB enzymes, which in turn suggests that the position of His134 could be more important for the Ni‐chelatase activity than that of His200. The SirB R134H/L200H variant showed lower activity than R134H, despite the four His residues found in SirB R134H/L200H. CD spectroscopy showed secondary structure denaturation and a slight difficulty in Ni‐binding of SirB R134H/L200H, which may be related to its lower activity. Finally, a docking simulation suggested that the His134 of the SirB R134H variant could function as a base catalyst for the Ni‐chelatase reaction in a class II chelatase architecture.

Keywords

• chelatase
• mutagenesis
• nickel
• porphyrin
• protein–ligand docking