Characterization of 3-Ketosteroid 9α-Hydroxylase, a Rieske Oxygenase in the Cholesterol Degradation Pathway of Mycobacterium tuberculosis

Capyk, Jenna K.; D’Angelo, I.; Strynadka, N.C.J.; Eltis, Lindsay D.

doi:10.1074/jbc.m900719200

Cited by 145 publications

(182 citation statements)

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“…Simi- larly, the apparent specificity of alkanesulfonate monooxygenase for alkanesulfonates was up to 112-fold greater (25). Nevertheless, the relative activity of HsaAB is very similar to that of its flanking enzymes in the cholesterol catabolic pathway, KshA (17) ), respectively, would help minimize intracellular concentrations of the latter, which is toxic to Mtb (6).…”

Section: Discussionmentioning

confidence: 99%

“…The extent to which side-chain and ring degradation occur concurrently is unclear. Ring degradation involves the successive actions of 3-ketosteroid-1⌬-dehydrogenase and 3-ketosteroid 9␣-hydroxylase, which result in the opening of ring B with concomitant aromatization of ring A to yield a phenol, 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3-HSA) (13)(14)(15)(16)(17). The phenolic ring of the latter has been predicted to be hydroxylated by HsaAB to yield a catechol, 3,4-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3,4-DHSA) ( Fig.…”

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confidence: 99%

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A Flavin-dependent Monooxygenase from Mycobacterium tuberculosis Involved in Cholesterol Catabolism

Dresen

Lin

D’Angelo

et al. 2010

Journal of Biological Chemistry

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102

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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-). However, 3,9-DHSA was a poorer substrate than 3-hy-). In the presence of 3-HSA the K mapp for O 2 was 100 ؎ 10 M. The crystal structure of HsaA to 2.5-Å 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°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. Mycobacterium tuberculosis (Mtb),6 the most devastating infectious agent of mortality worldwide (1), remains a primary public health threat due to synergy with the human immunodeficiency virus and the emergence of extensively drug-resistant strains. Mtb has the unusual ability to survive and replicate in the hostile environment of the macrophage (2, 3) utilizing largely unknown mechanisms. Genes essential to intracellular survival have been identified using genome-wide mutagenesis (4). A cluster of such genes was subsequently predicted to specify cholesterol catabolism (5). Targeted gene deletion studies subsequently indicated that cholesterol catabolism occurs throughout infection, contributing to the virulence of Mtb (6 -8). Although the exact role of cholesterol in infection remains unclear, the gene deletion studies are consistent with the high concentrations of cholesterol found within caseating granulomas and the observed congregation of bacteria around cholesterol foci (9).The cholesterol catabolic pathway of Mtb is very similar to that of other pathogenic and environmental actinomycetes (10), as initially predicted in Rhodococcus jostii RHA1 (5). Sidechain degradation is initiate...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

A Flavin-dependent Monooxygenase from Mycobacterium tuberculosis Involved in Cholesterol Catabolism

Dresen

Lin

D’Angelo

et al. 2010

Journal of Biological Chemistry

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102

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“…Several three-dimensional (3D) structures of ROs are currently available (3,4,6,7,8,9,13,15,17,19,20), including a single 3D structure of KshA, namely, that of M. tuberculosis H37Rv (Rv3526; KshA H37Rv ) (2). Although the protein sequences of ROs vary considerably, their tertiary structures are very similar overall.…”

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confidence: 99%

“…Characteristic for ROs, including KshA H37Rv , is the formation of trimers. These trimers either occur as ␣3 subunits (2,4,17,19) or may additionally include a smaller ␤ subunit of the oxygenase component, resulting in an (␣␤)3 enzyme complex (3,6,7,8,9,13,15,20). The active site of KshA H37Rv was shown to be composed of a ␤ sheet, including a loop region located at the entrance of the active site, flanked by two ␣ helices.…”

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confidence: 99%

“…9␣-Hydroxylated steroids are of particular interest for the synthesis of corticosteroids (14). KSH is a class IA monooxygenase, a two-component enzyme system comprised of the terminal oxygenase KshA, containing a Rieske Fe 2 S 2 cluster and a nonheme Fe 2ϩ located at the active site, and the reductase KshB, containing a plant-type Fe 2 S 2 cluster and the flavin cofactor flavin adenine dinucleotide (2,21,25). Interestingly, KshA and KshB both have been identified to be essential factors in the pathogenicity of Mycobacterium tuberculosis H37Rv (11).…”

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Structural Features in the KshA Terminal Oxygenase Protein That Determine Substrate Preference of 3-Ketosteroid 9 -Hydroxylase Enzymes

Petrusma

Dijkhuizen

Geize

2011

Journal of Bacteriology

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Rieske nonheme monooxygenase 3-ketosteroid 9␣-hydroxylase (KSH) enzymes play a central role in bacterial steroid catabolism. KSH is a two-component iron-sulfur-containing enzyme, with KshA representing the terminal oxygenase component and KshB the reductase component. We previously reported that the KshA1 and KshA5 homologues of Rhodococcus rhodochrous DSM43269 have clearly different substrate preferences. KshA protein sequence alignments and three-dimensional crystal structure information for KshA H37Rv of Mycobacterium tuberculosis H37Rv served to identify a variable region of 58 amino acids organized in a ␤ sheet that is part of the so-called helix-grip fold of the predicted KshA substrate binding pocket. Exchange of the ␤ sheets between KshA1 and KshA5 resulted in active chimeric enzymes with substrate preferences clearly resembling those of the donor enzymes. Exchange of smaller parts of the KshA1 and KshA5 ␤-sheet regions revealed that a highly variable loop region located at the entrance of the active site strongly contributes to KSH substrate preference. This loop region may be subject to conformational changes, thereby affecting binding of different substrates in the active site. This study provides novel insights into KshA structure-function relationships and shows that KSH monooxygenase enzymes are amenable to protein engineering for the development of biocatalysts with improved substrate specificities.

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3‐Ketosteroid‐9α‐Hydroxylase

Capyk

Strynadka

Eltis

2004

Handbook of Metalloproteins

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KshAB is a Rieske oxygenase (RO) in the aerobic steroid degradation pathways of bacteria. It is a monooxygenase responsible for the 9α‐hydroxylation of the nucleus of 3‐keto‐4‐ene steroids bearing short side chains at C17. It is composed of two domains arranged in a head‐to‐tail fashion to form the functional trimer. The N‐terminal Rieske domain harbors a Fe 2 S 2 cluster in which the two irons are coordinated by two cysteine and two histidine residues, respectively. The larger, C‐terminal catalytic domain contains a mononuclear nonheme iron coordinated by a His‐His‐Asp facial triad. Purified KshAB from Mycobacterium tuberculosis exhibits substrate specificities for 1,4‐androstadien‐3,17‐one and dioxygen 2‐3 orders of magnitude lower than those measured for other ROs and their best substrates. In agreement with the large fused‐ring substrate, the structure of KshA incorporates an active site channel and substrate binding pocket that are longer than those observed in other ROs, and positioned in a different relative orientation within the catalytic domain. KshA also possesses a C‐terminal helix which occupies a position not observed in other RO structures and which likely stabilizes trimeric quaternary structure. Finally, KshA exhibits a minimal core catalytic domain with respect to secondary structural elements that may prove archetypical for ROs.

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Characterization of 3-Ketosteroid 9α-Hydroxylase, a Rieske Oxygenase in the Cholesterol Degradation Pathway of Mycobacterium tuberculosis

Cited by 145 publications

References 59 publications

A Flavin-dependent Monooxygenase from Mycobacterium tuberculosis Involved in Cholesterol Catabolism

A Flavin-dependent Monooxygenase from Mycobacterium tuberculosis Involved in Cholesterol Catabolism

Structural Features in the KshA Terminal Oxygenase Protein That Determine Substrate Preference of 3-Ketosteroid 9 -Hydroxylase Enzymes

3‐Ketosteroid‐9α‐Hydroxylase

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