BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 248, 920–926 (1998) ARTICLE NO. RC988933 Ion Pairs Involved in Maintaining a Thermostable Structure of Glutamate Dehydrogenase from a Hyperthermophilic Archaeon Raja Noor Zaliha Abd. Rahman,* Shinsuke Fujiwara,† Haruki Nakamura,‡ Masahiro Takagi,† and Tadayuki Imanaka* ,1 *Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 606-8501, Japan; †Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; and ‡Department of Bioinformatics, Biomolecular Engineering Research Institute (BERI), 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan Received June 3, 1998 arises from the simultaneous effects of several forces, Intersubunit ion pairs are considered to be involved including hydrogen bonds which lead to decreased flex- for maintaining a stable structure of the glutamate de- ibility of the polypeptide chain (1, 13, 14). Several stud- hydrogenase (GDH) from hyperthermophiles. In order ies were performed on sequence comparison of meso- to demonstrate an effect of intersubunit ion pairs on philic and thermophilic enzymes of which three-dimen- the structural stability, two kinds of mutation (T138E, sional structures were available (1, 10, 15, 25). It has Thr at position 138 was replaced by Glu; E158Q, Glu been proposed that decreased flexibility and increased at position 158 was replaced by Gln) which add and hydrophobicity in a-helical regions of proteins are im- remove ion pairs, respectively, were introduced into portant in maintaining enzyme structure at high tem- Pk-gdhA gene encoding GDH from Pyrococcus koda- perature. In addition, ion pair formation seems im- karaensis KOD1. Addition of one ion pair (Pk-GDH- portant for maintaining the thermostable structures T138E) increased the optimum temperature and ther- (19). The three-dimensional structures of glutamate de- mostability. In contrast, Pk-GDH-E158Q showed lower hydrogenases (GDHs) from various organisms have optimum temperature and less thermostability than wild type GDH. Structure analysis of GDHs was per- shown that the number of probable surface ion pairs is formed by circular dichroism (CD) and indicated that increased in thermostable proteins (11, 23). Structural all recombinant enzymes (Pk-GDH, Pk-GDH-T138E, studies on indole-3-glycerolphosphate synthase (8), Pk-GDH-E158Q) possess different structures from that glyceraldehyde-3-phosphate dehydrogenase (12), alde- of natural GDH. Upon heat treatment (60°C, 2h), the hyde ferredoxin oxidoreductase (5) and rubredoxin (7), structures of Pk-GDH and Pk-GDH-T138E were con- also showed a large number of inter and intra subunit verted to another form close to the natural structure. ion pairs. However, no structural conversion by heat treatment Glutamate dehydrogenases (GDH) produces gluta- was observed in Pk-GDH-E158Q. These results indi- mate from 2-oxoglutarate and it functions as a major cate that intersubunit ion pairs play an important role assimilation enzyme in most hyperthermophilic ar- in forming thermostable structure of Pk-GDH.  1998 chaea (6, 20). The primary sequences of GDHs from Academic Press various organisms are available and have been used to classify GDHs into two different families: Family I (bacteria and lower eukarya) and Family II (archaea and higher eukarya) (3, 20). The alignment of the In recent years a considerable effort has been made to understand the mechanisms that determine the hexameric GDH sequences from hyperthermophilie thermal stability of proteins. Previous investigations against their mesophilic counterparts has shown that have indicated that the thermostability of a protein they display high similarity to each other (4, 23), al- though they exhibit clear differences in thermostabil- ity. The three dimensional structures of GDHs have 1 Corresponding author. Department of Synthetic Chemistry and been reported from three kinds of microorganisms (2, Biological Chemistry, Graduate School of Engineering, Kyoto Uni- 11, 28). The structural comparison of the two GDHs versity, Kyoto 606-8501, Japan. Fax: 81-(0)75-753-4703. E-mail: imanaka@sbchem.kyoto-u.ac.jp. from the hyperthermophiles Thermotoga maritima 0006-291X/98 $25.00 Copyright  1998 by Academic Press All rights of reproduction in any form reserved. 920