Refolding, characterization and crystal structure of (S)-malate dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernix

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Title
Refolding, characterization and crystal structure of (S)-malate dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernix
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Description

Tartrate oxidation activity was found in the crude extract of an aerobic hyperthermophilic archaeon Aeropyrum pernix, and the enzyme was identified as (S)-malate dehydrogenase (MDH), which, when produced in Escherichia coli, was mainly obtained as an inactive inclusion body. The inclusion body was dissolved in 6 M guanidine-HCl and gradually refolded to the active enzyme through dilution of the denaturant. The purified recombinant enzyme consisted of four identical subunits with a molecular mass of about 110 kDa. NADP was preferred as a coenzyme over NAD for (S)-malate oxidation and, unlike MDHs from other sources, this enzyme readily catalyzed the oxidation of (2S,3S)-tartrate and (2S,3R)-tartrate. The tartrate oxidation activity was also observed in MDHs from the hyperthermophilic archaea Methanocaldococcus jannaschii and Archaeoglobus fulgidus, suggesting these hyperthermophilic MDHs loosely bind their substrates. The refolded A. pernix MDH was also crystallized, and the structure was determined at a resolution of 2.9 A. Its overall structure was similar to those of the M. jannaschii, Chloroflexus aurantiacus, Chlorobium vibrioforme and Cryptosporidium parvum [lactate dehydrogenase-like] MDHs with root-mean-square-deviation values between 1.4 and 2.1 A. Consistent with earlier reports, Ala at position 53 was responsible for coenzyme specificity, and the next residue, Arg, was important for NADP binding. Structural comparison revealed that the hyperthermostability of the A. pernix MDH is likely attributable to its smaller cavity volume and larger numbers of ion pairs and ion-pair networks, but the molecular strategy for thermostability may be specific for each enzyme.

Author
著者 Kawakami Ryushi
著者(ヨミ) カワカミ リュウシ
著者(別表記) 川上 竜巳
著者 Sakuraba Haruhiko
著者(ヨミ) サクラバ ハルヒコ
著者(別表記) 櫻庭 春彦
著者 Goda Shuichiro
著者(ヨミ) ゴウダ シュウイチロウ
著者(別表記) 郷田 秀一郎
著者 Tsuge Hideaki
著者(ヨミ) ツゲ ヒデアキ
著者(別表記) 津下 英明
著者 Ohshima Toshihisa
著者(ヨミ) オオシマ トシヒサ
著者(別表記) 大島 敏久
Publication Title
Biochimica et Biophysica Acta
Publication Title Alternative
Biochim Biophys Acta.
Volume
1794
Issue
10
Start Page
1496
End Page
1504
Publisher
Elsevier
Published Date
200910
ISSN
0006-3002
NCID
AA00564635
PMID
19555779
DOI
10.1016/j.bbapap.2009.06.014
Resource Type
Journal Article
Language
eng
Resource URL
https://doi.org/10.1016/j.bbapap.2009.06.014
Rights
Copyright c 2009 Elsevier B.V.
この論文の本文は、「クリエイティブ・コモンズ 表示-非営利-改変禁止ライセンス 4.0 (CC-BY-NC-ND 4.0)」で提供されます。(https://creativecommons.org/licenses/by-nc-nd/4.0/deed.ja)。
The text of this paper is provided under the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (CC-BY-NC-ND 4.0).(https://creativecommons.org/licenses/by-nc-nd/4.0/).
Publisher's DOI: https://doi.org/10.1016/j.bbapap.2009.06.014
Text Version
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香川大学
Copyright (C) 2009 Kagawa University All rights reserved.