Steroid 5α-reductases (SRD5As), also known as 3-oxo-5α-steroid 4-dehydrogenases, are essential membrane-bound enzymes involved in steroid metabolism. Belonging to the NADPH-dependent oxidoreductase family, 5α-reductases catalyze steroids with 3-oxo-Δ4 structure, such as testosterone or progesterone, to produce their corresponding 3-oxo-5α steroids, which are necessary for a variety of physiological and pathological activities. Despite their significance, SRD5A structures are still in short supply to date. Here we describe a protocol for expression, purification, crystallization, structural determination, and functional analysis of PbSRD5A, the 5α-reductase from Proteobacteria bacterium sharing high sequence identity with human SRD5A1 and SRD5A2 isozymes, which we have recently structurally characterized using a lipidic cubic phase approach. Application of similar methods to other 5α-reductase isozymes will lead to breakthroughs in the understanding of the structure, function, and mechanism of oxidoreductases implicated in steroid metabolism.
Keywords: Bacterial SRD5A; In vitro biochemistry; Protein structure.
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