A sterol reductase responsible for the unusual 8(14)-unsaturation in bacterial sterol production and degradation

ABSTRACT Sterols are a class of lipids that play a crucial role in human health through their essential physiological roles and as a point of interaction between commensal and pathogenic bacteria. The biosynthesis and modification of these lipids is a well-characterized process in many eukaryotes and increasingly in bacteria. However, the proteins responsible for formation of the unusual 8(14)-unsaturation found in the sterols produced by aerobic methanotrophs, dinoflagellates, nematodes, and marine sponges, remains unknown. Here, we utilize a heterologous expression system to identify a bacterial 8,14-sterol reductase (8,14-Bsr) responsible for generating the 8(14)-unsaturation in the aerobic methanotroph Methylococcus capsulatus . This enzyme modifies the direct product of C-14 demethylation, reducing one double bond in the nuclear core structure and isomerizing the other to produce an 8(14)-sterol. We subsequently tested the requirement of putative active site residues for catalysis through site directed mutagenesis, identifying residues likely involved in interacting with the sterol substrate and directly catalyzing this reaction. Bioinformatic analysis of the distribution of 8,14-Bsr reveals it is unique to the bacterial domain, found primarily in the Methylococcaceae family , the Mycobacteriales order, and yet uncultured members of the Myxococcota phylum. Further phylogenetic analysis of 8,14-Bsr suggests it shares an evolutionary history with the C-14 demethylase in these organisms and that these two enzymes were likely inherited together. These results provide insight into novel sterol biochemistry, further delimiting sterol biosynthesis in the bacterial domain from eukaryotes and illustrating the importance of molecular characterization to identify bacterial proteins that interact with sterols. IMPORTANCE Many of the eukaryotic proteins required for the biosynthesis of sterols, such as cholesterol, have been characterized. However, the pathways governing analogous processes in the bacterial domain are less characterized. Here, we identify an 8,14-sterol reductase in aerobic methanotrophs. This enzyme carries out a unique biochemical reaction, saturating and isomerizing double bonds in the nuclear core structure to produce an 8(14)-sterol. This reductase is restricted to the bacterial domain, further separating the evolution of bacterial sterol production from eukaryotes. Additionally, we find this reductase is prevalent in members of the sterol degrading order Mycobacteriales, highlighting a potential role for this protein in the remodeling of host sterol production by these pathogens.