Psychrophilic valine dehydrogenase of the antarctic psychrophile, Cytophaga sp. KUC‐1
We found the occurrence of valine dehydrogenase in the cell extract of a psychrophilic bacterium, Cytophaga sp. KUC‐1, isolated from Antarctic seawater and purified the enzyme to homogeneity. The molecular mass of the enzyme was determined to be ≈ 154 kDa by gel filtration and that of the subunit wa...
Published in: | European Journal of Biochemistry |
---|---|
Main Authors: | , , , , |
Format: | Article in Journal/Newspaper |
Language: | English |
Published: |
Wiley
2001
|
Subjects: | |
Online Access: | http://dx.doi.org/10.1046/j.1432-1327.2001.02353.x https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1046%2Fj.1432-1327.2001.02353.x https://febs.onlinelibrary.wiley.com/doi/pdf/10.1046/j.1432-1327.2001.02353.x |
id |
crwiley:10.1046/j.1432-1327.2001.02353.x |
---|---|
record_format |
openpolar |
spelling |
crwiley:10.1046/j.1432-1327.2001.02353.x 2024-06-23T07:47:33+00:00 Psychrophilic valine dehydrogenase of the antarctic psychrophile, Cytophaga sp. KUC‐1 Purification, molecular characterization and expression Oikawa, Tadao Yamanaka, Kazuya Kazuoka, Takayuki Kanzawa, Noriyuki Soda, Kenji 2001 http://dx.doi.org/10.1046/j.1432-1327.2001.02353.x https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1046%2Fj.1432-1327.2001.02353.x https://febs.onlinelibrary.wiley.com/doi/pdf/10.1046/j.1432-1327.2001.02353.x en eng Wiley http://onlinelibrary.wiley.com/termsAndConditions#vor European Journal of Biochemistry volume 268, issue 16, page 4375-4383 ISSN 0014-2956 1432-1033 journal-article 2001 crwiley https://doi.org/10.1046/j.1432-1327.2001.02353.x 2024-06-13T04:25:13Z We found the occurrence of valine dehydrogenase in the cell extract of a psychrophilic bacterium, Cytophaga sp. KUC‐1, isolated from Antarctic seawater and purified the enzyme to homogeneity. The molecular mass of the enzyme was determined to be ≈ 154 kDa by gel filtration and that of the subunit was 43 kDa by SDS/PAGE: the enzyme was a homotetramer. The enzyme required NAD + as a coenzyme, and catalyzed the oxidative deamination of l ‐valine, l ‐isoleucine, l ‐leucine and the reductive amination of α‐ketoisovalerate, α‐ketovalerate, α‐ketoisocaproate, and α‐ketocaproate. The reaction proceeds through an iso‐ordered bi–bi mechanism. The enzyme was highly susceptible to heat treatment and the half‐life at 45 °C was estimated to be 2.4 min. The k cat / K m (µ& mgr −1 ·s −1 ) values for l ‐valine and NAD + at 20 °C were 27.48 and 421.6, respectively. The enzyme showed pro‐S stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of coenzyme. The gene encoding valine dehydrogenase was cloned into Escherichia coli (Novablue), and the primary structure of the enzyme was deduced on the basis of the nucleotide sequence of the gene encoding the enzyme. The enzyme contains 370 amino‐acid residues, and is highly homologous with S. coelicolor ValDH (identity, 46.7%) and S. fradiae ValDH (43.1%). Cytophaga sp. KUC‐1 ValDH contains much lower numbers of proline and arginine residues than those of other ValDHs. The changes probably lead to an increase in conformational flexibility of the Cytophaga enzyme molecule to enhance the catalytic activity at low temperatures. Article in Journal/Newspaper Antarc* Antarctic Wiley Online Library Antarctic The Antarctic European Journal of Biochemistry 268 16 4375 4383 |
institution |
Open Polar |
collection |
Wiley Online Library |
op_collection_id |
crwiley |
language |
English |
description |
We found the occurrence of valine dehydrogenase in the cell extract of a psychrophilic bacterium, Cytophaga sp. KUC‐1, isolated from Antarctic seawater and purified the enzyme to homogeneity. The molecular mass of the enzyme was determined to be ≈ 154 kDa by gel filtration and that of the subunit was 43 kDa by SDS/PAGE: the enzyme was a homotetramer. The enzyme required NAD + as a coenzyme, and catalyzed the oxidative deamination of l ‐valine, l ‐isoleucine, l ‐leucine and the reductive amination of α‐ketoisovalerate, α‐ketovalerate, α‐ketoisocaproate, and α‐ketocaproate. The reaction proceeds through an iso‐ordered bi–bi mechanism. The enzyme was highly susceptible to heat treatment and the half‐life at 45 °C was estimated to be 2.4 min. The k cat / K m (µ& mgr −1 ·s −1 ) values for l ‐valine and NAD + at 20 °C were 27.48 and 421.6, respectively. The enzyme showed pro‐S stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of coenzyme. The gene encoding valine dehydrogenase was cloned into Escherichia coli (Novablue), and the primary structure of the enzyme was deduced on the basis of the nucleotide sequence of the gene encoding the enzyme. The enzyme contains 370 amino‐acid residues, and is highly homologous with S. coelicolor ValDH (identity, 46.7%) and S. fradiae ValDH (43.1%). Cytophaga sp. KUC‐1 ValDH contains much lower numbers of proline and arginine residues than those of other ValDHs. The changes probably lead to an increase in conformational flexibility of the Cytophaga enzyme molecule to enhance the catalytic activity at low temperatures. |
format |
Article in Journal/Newspaper |
author |
Oikawa, Tadao Yamanaka, Kazuya Kazuoka, Takayuki Kanzawa, Noriyuki Soda, Kenji |
spellingShingle |
Oikawa, Tadao Yamanaka, Kazuya Kazuoka, Takayuki Kanzawa, Noriyuki Soda, Kenji Psychrophilic valine dehydrogenase of the antarctic psychrophile, Cytophaga sp. KUC‐1 |
author_facet |
Oikawa, Tadao Yamanaka, Kazuya Kazuoka, Takayuki Kanzawa, Noriyuki Soda, Kenji |
author_sort |
Oikawa, Tadao |
title |
Psychrophilic valine dehydrogenase of the antarctic psychrophile, Cytophaga sp. KUC‐1 |
title_short |
Psychrophilic valine dehydrogenase of the antarctic psychrophile, Cytophaga sp. KUC‐1 |
title_full |
Psychrophilic valine dehydrogenase of the antarctic psychrophile, Cytophaga sp. KUC‐1 |
title_fullStr |
Psychrophilic valine dehydrogenase of the antarctic psychrophile, Cytophaga sp. KUC‐1 |
title_full_unstemmed |
Psychrophilic valine dehydrogenase of the antarctic psychrophile, Cytophaga sp. KUC‐1 |
title_sort |
psychrophilic valine dehydrogenase of the antarctic psychrophile, cytophaga sp. kuc‐1 |
publisher |
Wiley |
publishDate |
2001 |
url |
http://dx.doi.org/10.1046/j.1432-1327.2001.02353.x https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1046%2Fj.1432-1327.2001.02353.x https://febs.onlinelibrary.wiley.com/doi/pdf/10.1046/j.1432-1327.2001.02353.x |
geographic |
Antarctic The Antarctic |
geographic_facet |
Antarctic The Antarctic |
genre |
Antarc* Antarctic |
genre_facet |
Antarc* Antarctic |
op_source |
European Journal of Biochemistry volume 268, issue 16, page 4375-4383 ISSN 0014-2956 1432-1033 |
op_rights |
http://onlinelibrary.wiley.com/termsAndConditions#vor |
op_doi |
https://doi.org/10.1046/j.1432-1327.2001.02353.x |
container_title |
European Journal of Biochemistry |
container_volume |
268 |
container_issue |
16 |
container_start_page |
4375 |
op_container_end_page |
4383 |
_version_ |
1802651671096983552 |