MUTATION AT A SINGLE ACIDIC AMINO-ACID ENHANCES THE HALOPHILIC BEHAVIOR OF MALATE-DEHYDROGENASE FROM HALOARCULA-MARISMORTUI IN PHYSIOLOGICAL SALTS

被引：59

作者：

MADERN, D

PFISTER, C

ZACCAI, G

机构：

[1] Institut de Biologie Structurale, Grenoble

来源：

EUROPEAN JOURNAL OF BIOCHEMISTRY | 1995年 / 230卷 / 03期

关键词：

PROTEIN STABILITY; HALOPHILES; SALT EFFECTS; HALOARCULA MARISMORTUI; ARCHAE;

D O I：

10.1111/j.1432-1033.1995.tb20659.x

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

In a statistical analysis of the amino acid compositions of 26 halophilic proteins, 24 showed an increase in acidic amino acids and a decrease in basic ones when compared to their non-halophlic homologues. The role of acidic residues in halophilic adaptation was investigated by site-directed mutagenesis of malate dehydrogenase (MalDH) from Haloarcula marismortui. In all of 40 non-halophilic homologous proteins, the position aligned with E243 in halophilic MalDH is occupied by a non-acidic amino acid, most frequently by arginine. The E243R mutant of halophilic MalDH was constructed, over-expressed in Escherichia coli, renatured and purified. Its salt-dependent catalytic activity was not affected compared to the wild-type enzyme and both proteins have the same K-m values for their substrates. The resistance to denaturation of the mutant was compared to that of the wild-type protein in different physiological salt (NaCl or KCl) and temperature conditions and interpreted in terms of classical quasi-thermodynamic parameters. The mutant is more halophilic than the wild-type protein; it is more sensitive to temperature and requires significantly higher concentrations of NaCl or KCl for equivalent stability. These results highlight the role of acidic amino acids in halophilic behaviour and are in agreement with a model in which these amino acids act cooperatively to organise hydrated ion binding to the protein.

引用

页码：1088 / 1095

页数：8

共 48 条

[1] Arndt E., Kroemer W., Hatakeyama T., Organisation and nucleotide sequence of a gene cluster coding for eight ribosomal proteins in the archaebacterium Halobacterium marismortui, J. Biol. Chem., 265, pp. 3034-3039, (1990)
[2] Baldacci G., Guinet F., Tillit J., Zaccai G., De Recondo A.M., Functional implications related to the gene structure of the elongation factor EF‐Tu from Halobacterium marismortui, Nucleic Acids Res., 18, pp. 507-511, (1990)
[3] Benachenhou N., Baldacci G., The genes for a halophilic glutamate dehydrogenase: sequence, transcription analysis and phylogenetic implications, Mol. & Gen. Genet., 230, pp. 345-352, (1991)
[4] Benachenhou-Lahfa N., Labedan B., Forterre P., PCR‐mediated cloning and sequencing of the gene encoding glutamate dehydrogenase from the archaeon Sulfolobus shibatae: identification of putative amino‐acid signatures for extremophilic adaptation, Gene, 140, pp. 17-24, (1994)
[5] Bohm G., Jaenicke R., Relevance of sequence statistics for the properties of extremophilic proteins, Int. J. Peptide Protein Res., 43, pp. 97-106, (1994)
[6] Bohm G., Jaenicke R., A structure‐based model for the halophilic adaptation dihydrofolate reductase from haloferax volcanii, "Protein Engineering, Design and Selection", 7, pp. 213-220, (1994)
[7] Bonnete F., Ebel C., Eisenberg H., Zaccai G., A biophysical study of halophilic malate dehydrogenase in solution: revised subunit structure and solvent interactions in native and recombinant enzyme, J. Chem. Soc. Faraday Trans., 89, pp. 2659-2666, (1993)
[8] Bonnete F., Madern D., Zaccai G., Stability against denaturation mechanisms in halophilic malate dehydrogenase “adapt” to solvent conditions, J. Mol. Biol., 244, pp. 436-447, (1994)
[9] Cendrin F., Chroboczek J., Zaccai G., Eisenberg H., Mevarech M., Cloning, sequencing and expression in Escherischia coli of the gene coding for malate dehydrogenase of the extremely halophilic archaebacterium Haloarcula marismortui, Biochemistry, 32, pp. 4308-4313, (1993)
[10] Conover R.K., Doolittle W.F., Characterization of a gene involved in histidine biosynthesis in Haloferax volcanii, J. Bacteriol., 172, pp. 3244-3249, (1990)

← 1 2 3 4 5 →