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Title: Crystallization, X-ray diffraction analysis and phasing of 17β-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus

Abstract

The expression, purification and crystallization of 17β-hydroxysteroid dehydrogenase from the filamentous fungus C. lunatus and its Y167F mutant, both in the apo form, are described. X-ray diffraction analysis and phasing by Patterson-search techniques are reported. 17β-Hydroxysteroid dehydrogenase from the filamentous fungus Cochliobolus lunatus (17β-HSDcl) is an NADP(H)-dependent enzyme that preferentially catalyses the oxidoreduction of oestrogens and androgens. The enzyme belongs to the short-chain dehydrogenase/reductase superfamily and is the only fungal hydroxysteroid dehydrogenase known to date. 17β-HSDcl has recently been characterized and cloned and has been the subject of several functional studies. Although several hypotheses on the physiological role of 17β-HSDcl in fungal metabolism have been formulated, its function is still unclear. An X-ray crystallographic study has been undertaken and the optimal conditions for crystallization of 17β-HSDcl (apo form) were established, resulting in well shaped crystals that diffracted to 1.7 Å resolution. The space group was identified as I4{sub 1}22, with unit-cell parameters a = b = 67.14, c = 266.77 Å. Phasing was successfully performed by Patterson search techniques. A catalytic inactive mutant Tyr167Phe was also engineered, expressed, purified and crystallized for functional and structural studies.

Authors:
 [1]; ; ; ;  [2];  [1]
  1. Institute of Crystallography, CNR, Trieste Outstation, Area Science Park-Basovizza, S.S.14, Km 163.5, I-34012 Trieste (Italy)
  2. Institute of Biochemistry, Medical Faculty, Vrazov trg 2, SI-1000 Ljubljana (Slovenia)
Publication Date:
OSTI Identifier:
22356207
Resource Type:
Journal Article
Journal Name:
Acta Crystallographica. Section F
Additional Journal Information:
Journal Volume: 61; Journal Issue: Pt 12; Other Information: PMCID: PMC1978160; PMID: 16511227; PUBLISHER-ID: za5117; OAI: oai:pubmedcentral.nih.gov:1978160; Copyright (c) International Union of Crystallography 2005; Country of input: International Atomic Energy Agency (IAEA); Journal ID: ISSN 1744-3091
Country of Publication:
United Kingdom
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; CRYSTALLIZATION; CRYSTALS; RESOLUTION; SPACE GROUPS; X-RAY DIFFRACTION

Citation Formats

Cassetta, Alberto, Büdefeld, Tomaž, Lanišnik Rižner, Tea, Kristan, Katja, Stojan, Jure, and Lamba, Doriano. Crystallization, X-ray diffraction analysis and phasing of 17β-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus. United Kingdom: N. p., 2005. Web. doi:10.1107/S1744309105034949.
Cassetta, Alberto, Büdefeld, Tomaž, Lanišnik Rižner, Tea, Kristan, Katja, Stojan, Jure, & Lamba, Doriano. Crystallization, X-ray diffraction analysis and phasing of 17β-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus. United Kingdom. https://doi.org/10.1107/S1744309105034949
Cassetta, Alberto, Büdefeld, Tomaž, Lanišnik Rižner, Tea, Kristan, Katja, Stojan, Jure, and Lamba, Doriano. Thu . "Crystallization, X-ray diffraction analysis and phasing of 17β-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus". United Kingdom. https://doi.org/10.1107/S1744309105034949.
@article{osti_22356207,
title = {Crystallization, X-ray diffraction analysis and phasing of 17β-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus},
author = {Cassetta, Alberto and Büdefeld, Tomaž and Lanišnik Rižner, Tea and Kristan, Katja and Stojan, Jure and Lamba, Doriano},
abstractNote = {The expression, purification and crystallization of 17β-hydroxysteroid dehydrogenase from the filamentous fungus C. lunatus and its Y167F mutant, both in the apo form, are described. X-ray diffraction analysis and phasing by Patterson-search techniques are reported. 17β-Hydroxysteroid dehydrogenase from the filamentous fungus Cochliobolus lunatus (17β-HSDcl) is an NADP(H)-dependent enzyme that preferentially catalyses the oxidoreduction of oestrogens and androgens. The enzyme belongs to the short-chain dehydrogenase/reductase superfamily and is the only fungal hydroxysteroid dehydrogenase known to date. 17β-HSDcl has recently been characterized and cloned and has been the subject of several functional studies. Although several hypotheses on the physiological role of 17β-HSDcl in fungal metabolism have been formulated, its function is still unclear. An X-ray crystallographic study has been undertaken and the optimal conditions for crystallization of 17β-HSDcl (apo form) were established, resulting in well shaped crystals that diffracted to 1.7 Å resolution. The space group was identified as I4{sub 1}22, with unit-cell parameters a = b = 67.14, c = 266.77 Å. Phasing was successfully performed by Patterson search techniques. A catalytic inactive mutant Tyr167Phe was also engineered, expressed, purified and crystallized for functional and structural studies.},
doi = {10.1107/S1744309105034949},
url = {https://www.osti.gov/biblio/22356207}, journal = {Acta Crystallographica. Section F},
issn = {1744-3091},
number = Pt 12,
volume = 61,
place = {United Kingdom},
year = {2005},
month = {12}
}