The catalytic roles of P185 and T188 and substrate-binding loop flexibility in 3α-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni.

3α-Hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni reversibly catalyzes the oxidation of androsterone with NAD(+) to form androstanedione and NADH. Structurally the substrate-binding loop of the residues, T188-K208, is unresolved, while binding with NAD(+) causes the appe...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Chi-Ching Hwang, Yi-Hsun Chang, Hwei-Jen Lee, Tzu-Pin Wang, Yu-Mei Su, Hsin-Wei Chen, Po-Huang Liang
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/5d21877c0bad475fb497996253eccd6d
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:5d21877c0bad475fb497996253eccd6d
record_format	dspace
spelling	oai:doaj.org-article:5d21877c0bad475fb497996253eccd6d2021-11-18T07:44:26ZThe catalytic roles of P185 and T188 and substrate-binding loop flexibility in 3α-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni.1932-620310.1371/journal.pone.0063594https://doaj.org/article/5d21877c0bad475fb497996253eccd6d2013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23717450/pdf/?tool=EBIhttps://doaj.org/toc/1932-62033α-Hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni reversibly catalyzes the oxidation of androsterone with NAD(+) to form androstanedione and NADH. Structurally the substrate-binding loop of the residues, T188-K208, is unresolved, while binding with NAD(+) causes the appearance of T188-P191 in the binary complex. This study determines the functional roles of the flexible substrate-binding loop in conformational changes and enzyme catalysis. A stopped-flow study reveals that the rate-limiting step in the reaction is the release of the NADH. The mutation at P185 in the hinge region and T188 in the loop causes a significant increase in the Kd value for NADH by fluorescence titration. A kinetic study of the mutants of P185A, P185G, T188A and T188S shows an increase in k(cat), K(androsterone) and K(iNAD) and equal primary isotope effects of (D)V and (D) (V/K). Therefore, these mutants increase the dissociation of the nucleotide cofactor, thereby increasing the rate of release of the product and producing the rate-limiting step in the hydride transfer. Simulated molecular modeling gives results that are consistent with the conformational change in the substrate-binding loop after NAD(+) binding. These results indicate that P185, T188 and the flexible substrate-binding loop are involved in binding with the nucleotide cofactor and with androsterone and are also involved in catalysis.Chi-Ching HwangYi-Hsun ChangHwei-Jen LeeTzu-Pin WangYu-Mei SuHsin-Wei ChenPo-Huang LiangPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 5, p e63594 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Chi-Ching Hwang Yi-Hsun Chang Hwei-Jen Lee Tzu-Pin Wang Yu-Mei Su Hsin-Wei Chen Po-Huang Liang The catalytic roles of P185 and T188 and substrate-binding loop flexibility in 3α-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni.
description	3α-Hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni reversibly catalyzes the oxidation of androsterone with NAD(+) to form androstanedione and NADH. Structurally the substrate-binding loop of the residues, T188-K208, is unresolved, while binding with NAD(+) causes the appearance of T188-P191 in the binary complex. This study determines the functional roles of the flexible substrate-binding loop in conformational changes and enzyme catalysis. A stopped-flow study reveals that the rate-limiting step in the reaction is the release of the NADH. The mutation at P185 in the hinge region and T188 in the loop causes a significant increase in the Kd value for NADH by fluorescence titration. A kinetic study of the mutants of P185A, P185G, T188A and T188S shows an increase in k(cat), K(androsterone) and K(iNAD) and equal primary isotope effects of (D)V and (D) (V/K). Therefore, these mutants increase the dissociation of the nucleotide cofactor, thereby increasing the rate of release of the product and producing the rate-limiting step in the hydride transfer. Simulated molecular modeling gives results that are consistent with the conformational change in the substrate-binding loop after NAD(+) binding. These results indicate that P185, T188 and the flexible substrate-binding loop are involved in binding with the nucleotide cofactor and with androsterone and are also involved in catalysis.
format	article
author	Chi-Ching Hwang Yi-Hsun Chang Hwei-Jen Lee Tzu-Pin Wang Yu-Mei Su Hsin-Wei Chen Po-Huang Liang
author_facet	Chi-Ching Hwang Yi-Hsun Chang Hwei-Jen Lee Tzu-Pin Wang Yu-Mei Su Hsin-Wei Chen Po-Huang Liang
author_sort	Chi-Ching Hwang
title	The catalytic roles of P185 and T188 and substrate-binding loop flexibility in 3α-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni.
title_short	The catalytic roles of P185 and T188 and substrate-binding loop flexibility in 3α-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni.
title_full	The catalytic roles of P185 and T188 and substrate-binding loop flexibility in 3α-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni.
title_fullStr	The catalytic roles of P185 and T188 and substrate-binding loop flexibility in 3α-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni.
title_full_unstemmed	The catalytic roles of P185 and T188 and substrate-binding loop flexibility in 3α-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni.
title_sort	catalytic roles of p185 and t188 and substrate-binding loop flexibility in 3α-hydroxysteroid dehydrogenase/carbonyl reductase from comamonas testosteroni.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/5d21877c0bad475fb497996253eccd6d
work_keys_str_mv	AT chichinghwang thecatalyticrolesofp185andt188andsubstratebindingloopflexibilityin3ahydroxysteroiddehydrogenasecarbonylreductasefromcomamonastestosteroni AT yihsunchang thecatalyticrolesofp185andt188andsubstratebindingloopflexibilityin3ahydroxysteroiddehydrogenasecarbonylreductasefromcomamonastestosteroni AT hweijenlee thecatalyticrolesofp185andt188andsubstratebindingloopflexibilityin3ahydroxysteroiddehydrogenasecarbonylreductasefromcomamonastestosteroni AT tzupinwang thecatalyticrolesofp185andt188andsubstratebindingloopflexibilityin3ahydroxysteroiddehydrogenasecarbonylreductasefromcomamonastestosteroni AT yumeisu thecatalyticrolesofp185andt188andsubstratebindingloopflexibilityin3ahydroxysteroiddehydrogenasecarbonylreductasefromcomamonastestosteroni AT hsinweichen thecatalyticrolesofp185andt188andsubstratebindingloopflexibilityin3ahydroxysteroiddehydrogenasecarbonylreductasefromcomamonastestosteroni AT pohuangliang thecatalyticrolesofp185andt188andsubstratebindingloopflexibilityin3ahydroxysteroiddehydrogenasecarbonylreductasefromcomamonastestosteroni AT chichinghwang catalyticrolesofp185andt188andsubstratebindingloopflexibilityin3ahydroxysteroiddehydrogenasecarbonylreductasefromcomamonastestosteroni AT yihsunchang catalyticrolesofp185andt188andsubstratebindingloopflexibilityin3ahydroxysteroiddehydrogenasecarbonylreductasefromcomamonastestosteroni AT hweijenlee catalyticrolesofp185andt188andsubstratebindingloopflexibilityin3ahydroxysteroiddehydrogenasecarbonylreductasefromcomamonastestosteroni AT tzupinwang catalyticrolesofp185andt188andsubstratebindingloopflexibilityin3ahydroxysteroiddehydrogenasecarbonylreductasefromcomamonastestosteroni AT yumeisu catalyticrolesofp185andt188andsubstratebindingloopflexibilityin3ahydroxysteroiddehydrogenasecarbonylreductasefromcomamonastestosteroni AT hsinweichen catalyticrolesofp185andt188andsubstratebindingloopflexibilityin3ahydroxysteroiddehydrogenasecarbonylreductasefromcomamonastestosteroni AT pohuangliang catalyticrolesofp185andt188andsubstratebindingloopflexibilityin3ahydroxysteroiddehydrogenasecarbonylreductasefromcomamonastestosteroni
_version_	1718423038174691328

The catalytic roles of P185 and T188 and substrate-binding loop flexibility in 3α-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni.

Ejemplares similares