Functional diversification within the heme-binding split-barrel family

Authors: GROSJEAN, NICHOLAS - Brookhaven National Laboratory; ZHANG, LIFANG - Cold Spring Harbor Laboratory; KUMARAN, DESIGAN - Brookhaven National Laboratory; XIE, MENG - Brookhaven National Laboratory; FAHEY, AUDREY - Cold Spring Harbor Laboratory; SANTIAGO, KASSANDRA - Brookhaven National Laboratory; HU, FANGLE - Cold Spring Harbor Laboratory; REGULSKI, MICHAEL - Cold Spring Harbor Laboratory; BLABY, IAN - Lawrence Berkeley National Laboratory; BLABY-HAAS, CRYSTEN - Lawrence Berkeley National Laboratory; Ware, Doreen

Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/10/2024
Publication Date: N/A
Citation: N/A

Interpretive Summary: Iron (Fe) is one of the most important micronutrients. Fe is loaded into heme, which is an important carrier for the transfer of oxygen. However, free heme can react with lipoproteins and oxygen species, causing damage. Therefore, the biosynthesis and turnover of heme must be tightly regulated. In this work, we present the characterization of a large family which shares an FMN-binding split barrel domain functioning as a heme-binding protein, using various approaches including sequence clustering, phylogenetic analysis, and domain analysis. One group, named HOZ (homolog of HugZ), is highly conserved in prokaryotes, plants, and algae. In the plant Arabidopsis there are two HOZ subfamilies, HOZ1 and HOZ2. Disruption of HOZ1 and HOZ2A causes developmental delays.

Technical Abstract: Due to neofunctionalization, a single fold can be identified in multiple proteins that have distinct molecular functions. Depending on the time that has passed since gene duplication and the number of mutations, the sequence similarity between functionally divergent proteins can be relatively high, eroding the value of sequence similarity as the sole tool for accurately annotating the function of uncharacterized homologs. Here, we combine bioinformatic approaches with targeted experimentation and revedal a large multi-functional family of putative enzymatic and non-enzymatic proteins involved in heme metabolism. This family (homolog of HugZ (HOZ)) is embedded in the “FMN-binding split barrel” superfamily, where amino acid mutation combined with independent instances of domain fusion in prokaryotes, plants, and algae have resulted in separate groups of proteins that bind heme and either catalyze its degradation or function as non-enzymatic heme sensors. In prokaryotes, these proteins are often involved in iron assimilation, several plant and algal homologs are predicted to degrade heme in the plastid or regulate heme biosynthesis. In the plant Arabidopsis thaliana, which contains two HOZ subfamilies that can degrade heme in vitro (HOZ1 and HOZ2), disruption of AtHOZ1 (AT3G03890) or AtHOZ2A(AT1G51560) causes developmental delays, pointing to important biological roles in the plastid. In the tree Populus trichocarpa, a recent duplication event of PtHOZ1 has resulted in localization to the cytosol. Structural characterization of this cytosolic paralog reveals a previously unknown metal-binding site. This study unifies our understanding of the sequence-structure-function relationships within this multi-lineage family of heme-binding proteins and presents new molecular players in plant and bacterial heme metabolism.