3AFF

Crystal structure of the HsaA monooxygenase from M. tuberculosis

3AFF の概要

• エントリーDOI: 10.2210/pdb3aff/pdb
• 関連するPDBエントリー: 3AFE
• 分子名称: Hydroxylase, putative (2 entities in total)
• 機能のキーワード: hsaa, cholesterol 3hsa monooxygenase, oxidoreductase
• 由来する生物種: Mycobacterium tuberculosis
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 86386.89

構造登録者

D'Angelo, I.,Lin, L.Y.,Dresen, C.,Tocheva, E.I.,Strynadka, N.,Eltis, L.D. (登録日: 2010-02-28, 公開日: 2010-05-26, 最終更新日: 2023-11-01)

主引用文献

Dresen, C.,Lin, L.Y.,D'Angelo, I.,Tocheva, E.I.,Strynadka, N.,Eltis, L.D.
A flavin-dependent monooxygenase from Mycobacterium tuberculosis involved in cholesterol catabolism
J.Biol.Chem., 285:22264-22275, 2010

PubMed Abstract: Mycobacterium tuberculosis (Mtb) and Rhodococcus jostii RHA1 have similar cholesterol catabolic pathways. This pathway contributes to the pathogenicity of Mtb. The hsaAB cholesterol catabolic genes have been predicted to encode the oxygenase and reductase, respectively, of a flavin-dependent mono-oxygenase that hydroxylates 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3-HSA) to a catechol. An hsaA deletion mutant of RHA1 did not grow on cholesterol but transformed the latter to 3-HSA and related metabolites in which each of the two keto groups was reduced: 3,9-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-17-one (3,9-DHSA) and 3,17-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9-one (3,17-DHSA). Purified 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione 4-hydroxylase (HsaAB) from Mtb had higher specificity for 3-HSA than for 3,17-DHSA (apparent k(cat)/K(m) = 1000 +/- 100 M(-1) s(-1) versus 700 +/- 100 M(-1) s(-1)). However, 3,9-DHSA was a poorer substrate than 3-hydroxybiphenyl (apparent k(cat)/K(m) = 80 +/- 40 M(-1) s(-1)). In the presence of 3-HSA the K(m)(app) for O(2) was 100 +/- 10 microM. The crystal structure of HsaA to 2.5-A resolution revealed that the enzyme has the same fold, flavin-binding site, and catalytic residues as p-hydroxyphenyl acetate hydroxylase. However, HsaA has a much larger phenol-binding site, consistent with the enzyme's substrate specificity. In addition, a second crystal form of HsaA revealed that a C-terminal flap (Val(367)-Val(394)) could adopt two conformations differing by a rigid body rotation of 25 degrees around Arg(366). This rotation appears to gate the likely flavin entrance to the active site. In docking studies with 3-HSA and flavin, the closed conformation provided a rationale for the enzyme's substrate specificity. Overall, the structural and functional data establish the physiological role of HsaAB and provide a basis to further investigate an important class of monooxygenases as well as the bacterial catabolism of steroids.

PubMed: 20448045
DOI: 10.1074/jbc.M109.099028

実験手法

X-RAY DIFFRACTION (2 Å)