Structural investigations of the ferredoxin and terminal oxygenase components of the biphenyl 2,3-dioxygenase from Sphingobium yanoikuyae B1

Ferraro, D.J.; Brown, Eric N.; Yu, Chung Huang; Parales, Rebecca E.; Gibson, David T.; Ramaswamy, S.

doi:10.1186/1472-6807-7-10

Cited by 86 publications

(74 citation statements)

References 59 publications

(89 reference statements)

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“…Bean-shaped residual density was observed for the exogenous ligand prior to its inclusion in the model. This is similar to what has been observed in structures of other ROs (52)(53)(54). When this density was modeled as a single fully occupied solvent species, the temperature factor of the refined ligand (34 Å 2 ) was comparable with those of surrounding protein atoms.…”

supporting

confidence: 83%

“…Finally, a tentative refinement with two fully occupied solvent species resulted in a distance between them of 1.9 Å, with positive residual density present whenever occupancy of either or both species was reduced. Overall, the observable density probably reflects the presence of multiple species with partial occupancies, as has been postulated for other ROs (52)(53)(54).…”

supporting

confidence: 58%

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

Capyk

D’Angelo

Strynadka

et al. 2009

Journal of Biological Chemistry

145

175

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KshAB (3-Ketosteroid 9␣-hydroxylase) is a two-component Rieske oxygenase (RO) in the cholesterol catabolic pathway of Mycobacterium tuberculosis. Although the enzyme has been implicated in pathogenesis, it has largely been characterized by bioinformatics and molecular genetics. Purified KshB, the reductase component, was a monomeric protein containing a plant-type [2Fe-2S] cluster and FAD. KshA, the oxygenase, was a homotrimer containing a Rieske [2Fe-2S] cluster and mononuclear ferrous iron. Of two potential substrates, reconstituted KshAB had twice the specificity for 1,4-androstadiene-3,17-dione as for 4-androstene-3,17-dione. The transformation of both substrates was well coupled to the consumption of O 2 . Nevertheless, the reactivity of KshAB with O 2 was low in the presence of 1,4-androstadiene-3,17-dione, with a k cat / K mO 2 of 2450 ؎ 80 M ؊1 s ؊1. The crystallographic structure of KshA, determined to 2.3 Å , revealed an overall fold and a head-to-tail subunit arrangement typical of ROs. The central fold of the catalytic domain lacks all insertions found in characterized ROs, consistent with a minimal and perhaps archetypical RO catalytic domain. The structure of KshA is further distinguished by a C-terminal helix, which stabilizes subunit interactions in the functional trimer. Finally, the substrate-binding pocket extends farther into KshA than in other ROs, consistent with the large steroid substrate, and the funnel accessing the active site is differently orientated. This study provides a solid basis for further studies of a key steroidtransforming enzyme of biotechnological and medical importance.Mycobacterium tuberculosis, arguably the world's most successful pathogen, infects one-third of the human population and has again become a global threat due in part to the emergence of extensively drug-resistant strains (XDR-TB) that are virtually untreatable with current medicines (1). Despite this alarming development, a surprising amount of the pathogen's physiology remains unknown. One recently discovered aspect of the physiology of M. tuberculosis is its cholesterol catabolic pathway (2). Studies of mutants in cholesterol uptake (3) and degradation (4) in various animal models have indicated that cholesterol catabolism is most important during the chronic phase of infection, although the latter study also provided evidence that it occurs from an early stage and contributes to dissemination of the pathogen in the host. Further study of cholesterol catabolism and the pathway enzymes are required to elucidate the precise role of cholesterol catabolism in infection.The cholesterol catabolic pathway of M. tuberculosis involves degradation of the branched alkyl side chain and the four-ringed steroid nucleus, as occurs in Rhodococcus jostii RHA1, a nonpathogenic, mycolic acid-producing actinomycete (2), although it is unclear whether the order of this degradation is obligatory. Side-chain degradation proceeds via a ␤-oxidative type process. Degradation of the steroid nucleus is initiated by 3␤-hydroxysteroi...

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supporting

confidence: 83%

supporting

confidence: 58%

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

Capyk

D’Angelo

Strynadka

et al. 2009

Journal of Biological Chemistry

145

175

View full text Add to dashboard Cite

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“…Similar enlargement of the substratebinding pocket has been reported in other RO oxygenase components to accommodate larger substrates, such as benzo[a]pyrene, benzo[a]anthracene, and chrysene. 19,20) His183, His187, and Asp333 coordinate the ferrous iron active site, 10) forming a 2-His-1-carboxylate facial triad (Fig. 4A).…”

Section: Discussionmentioning

confidence: 99%

Alteration of the Substrate Specificity of the Angular Dioxygenase Carbazole 1,9a-Dioxygenase

Uchimura

Horisaki

Umeda

et al. 2008

Bioscience, Biotechnology, and Biochemistry

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“…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.…”

mentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

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.

show abstract

Structural investigations of the ferredoxin and terminal oxygenase components of the biphenyl 2,3-dioxygenase from Sphingobium yanoikuyae B1

Cited by 86 publications

References 59 publications

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

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

Alteration of the Substrate Specificity of the Angular Dioxygenase Carbazole 1,9a-Dioxygenase

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

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