3-Ketosteroid-Delta (5-Alpha) Dehydrogenase
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The enzyme 3-ketosteroid-Delta-4-(5-alpha)dehydrogenase reduces testosterone to dihydrotesterone, which is the metabolite for the formation of male genitalia. A mutation of this metabolite results in the genetic disorder 5-alpha reductase deficiency. This is a rare autosomal recessive disease which is sex linked, as it is mostly found in genetic males. This disease is most prominent in the Dominican Republic, Turkey, and New Guinea, where there is a decrease in the gene pool due to increased levels of inter-familial reproduction.
Full backbone structureTwo-stranded antiparallel beta sheets connect the two domains within the protien. Domain F contains a non-covalently bound FAD cofactor; it also consists of a central 5-stranded parallel beta sheet marked on one side by four additional beta strands. On the other side, it is marked by three alpha helices. The substrate-binding domain (S) inserts into Domain F, creating two parts (F1 and F2). The S Domain consists of four-stranded antiparallel Beta sheets marked on both sides by two alpha helices. The active site is located at the interface between the F and S domains next to the isoalloxazine ring system of the FAD cofactor. The F domain will be depicted with orange color and the S domain will be depicted by the purple color. The alpha helices are colored pink and the beta sheets are colored red for easy distinction.
F and S DomainsThe FAD binding site is non-covalently bonded in a long cleft within the F-domain. The ADP portion of the FAD molecule is bound by a motif resembling the beta-alpha-beta-alpha-beta dinucleotide binding motif, which is frequently observed in flavoprotein. The adenine is bound by two hydrogen bonds to the backbone amide and carbonyl group of Val-205. The two hydroxyl groups of the adenosine ribose are hydrogen bonded to Glu-51. the two phosphate groups have hydrogen bonding interactions with the backbone amides of Ile-31, Ala-32, Ala-59, Thr-60, and Arg-456 (shown below in flashing green). There is also a salt bridge between one of the phosphates and His-268
FAD Binding SiteThe isoalloxazine ring system is located near the interface of the F and S domains. The isoalloxazine site is conducive to maintaining the stability of bound active site. The N5 nitrogen atom is hydrogen bonded to the backbone amide of Gly-64 and the main chain NHs of Ser-471(blue) and Leu-472 (blue) have hydrogen bonding interaction with the oxygen of the prymidine ring. Ser-471 and Leu-472 are at the N-terminal end of the long C-terminal alpha helix, formed by residues 470-489 (red), and dipole moment provided by this helix may stabilize the negative charge on the oxygen atom in the anionic form of FAD.
Isoalloxazine Ring SystemThe active site is open to the solvent and contains a well-ordered network of water molecules and two acetate ions (black). A chloride ion (green) is located behind the dimethyl benzene ring of the isoalloxazine ring system. The ligand is 4-androstene-3, 17-dione, and it binds to the isoalloxazine ring system, causing the displacement of three water molecules and two acetate molecules.
The Active SiteFlavoproteins involved in the dehydrogenation reactions often display a few recurrent features. In many flavoproteins, catalysis takes place at a location that is shielded from the solvent. This enhances the strength of the polar interactions, making this a fundamental process of substrate activation. Tyr466 (grey) is assumed to act as the catalytic acid, and ser468 grey) acts as the catalytic base.
Site of CatalysisThe mutations that make this enzyme not function properly in the metabolism of testosterone can be seen below in the areas of flashing black. One is a heterozygous missense mutation at codon 123, convertin GGA to AGA. Another common mutation is a frameshift mutation at amino acid 251, adding 23 additional amino acids to the C-terminal. Also, insertion mutations of a TA at amino acid 188 and 189 occur, that lead to the premature termination of the protein. Finally, Mutations to Tyr 466 result in a loss of catalysis to the enzyme.
MutationsVan Oosterwijk, N., Knol, J., Dijkhuizen, L., Van der Geize, R., Dijkstra, B. (2012). Protein structure and folding: Structure and Catalytic Mechanism of 3-Ketosteroid-delta4-5alpha-dehydrogenase from Rhodococcus jostii RHA1 genone. Journal of Biological Chemistry. 287: 30975-30983, DOI: 10.1074/jbc.M112.374306
Mechanism Medicine. Testosterone Production. retrieved from: http://www.youtube.com/watch?v=djqqao2Uebo.