Amino acid residues involved in cold adaptation of isocitrate lyase from a psychrophilic bacterium, Colwellia maris.
نویسندگان
چکیده
To investigate the mechanism of cold adaptation of isocitrate lyase (ICL; EC 4.1.3.1) from the psychrophilic bacterium Colwellia maris, Gln207 and Gln217 of this enzyme were substituted by His and Lys, respectively, by site-directed mutagenesis. His184 and Lys194 of ICL from Escherichia coli, corresponding to the two Gln residues of C. maris ICL, are highly conserved in the ICLs of many organisms and are known to be essential for catalytic function. The mutated ICLs (Cm-Q207H and Cm-Q217K, respectively) and wild-type enzymes of C. maris and E. coli (Cm-WT and Ec-WT) with His-tagged peptides were overexpressed in E. coli cells and purified to homogeneity. Thermolabile Cm-WT and mutated ICLs were susceptible to digestion with trypsin, while relatively thermostable Ec-WT was resistant to trypsin digestion, suggesting that the thermostability and resistance to tryptic digestion of the ICLs are related. Cm-Q207H and Cm-Q217K showed specific activities similar to Cm-WT at temperatures between 30 degrees C and 40 degrees C, but their activities between 10 degrees C and 25 degrees C were decreased, indicating that the two Gln residues of the C. maris ICL play important roles in its cold adaptation. Phylogenetic analysis of ICLs from various organisms revealed that the C. maris ICL can be categorized in a novel group, subfamily 3, together with several eubacterial ICLs.
منابع مشابه
Analysis of amino acid residues involved in cold activity of monomeric isocitrate dehydrogenase from psychrophilic bacteria, Colwellia maris and Colwellia psychrerythraea.
Monomeric isocitrate dehydrogenases from psychrophilic bacteria, Colwellia maris and Colwellia psychrerythraea (CmIDH-II and CpIDH-M, respectively) are cold-adapted enzymes and show a high degree of amino acid sequential identity to each other (77%). However, maximum activity of CpIDH-M at optimum temperature is much less than that of CmIDH-II. In the C-terminal region 3 of these enzymes, which...
متن کاملAnalysis of the amino acid residues involved in the thermal properties of the monomeric isocitrate dehydrogenases of the psychrophilic bacterium Colwellia maris and the mesophilic bacterium Azotobacter vinelandii.
Cold-adapted monomeric isocitrate dehydrogenase of a psychrophilic bacterium, Colwellia maris, (CmIDH) showed a high degree of amino acid sequential identity (69.5%) to a mesophilic nitrogen-fixing bacterium, Azotobacter vinelandii, (AvIDH). In this study, three Ala residues of CmIDH and the corresponding Pro residues of AvIDH were exchanged by site-directed mutagenesis, and several properties ...
متن کاملElucidation of stability determinants of cold-adapted monomeric isocitrate dehydrogenase from a psychrophilic bacterium, Colwellia maris, by construction of chimeric enzymes.
To elucidate determinants of differences in thermostability between mesophilic and psychrophilic monomeric isocitrate dehydrogenases (IDHs) from Azotobacter vinelandii (AvIDH) and Colwellia maris (CmIDH), respectively, chimeric enzymes derived from the two IDHs were constructed based on the recently resolved three-dimensional structure of AvIDH, and several characteristics of the two wild-type ...
متن کاملAssignment of Vibrio sp. strain ABE-1 to Colwellia maris sp. nov., a new psychrophilic bacterium.
A psychrophilic bacterium, previously described as Vibrio sp. strain ABE-1T, has been reassigned by phenotypic characterization, chemotaxonomic analysis and 16S rRNA phylogenetic analysis. The organism was curved rods and it could reduce nitrate to nitrite and hydrolyse gelatin and DNA, but not chitin. NaCl was required for growth. This strain was susceptible to the vibriostatic compound O/129....
متن کاملCrystal structure of UbiX, an aromatic acid decarboxylase from the psychrophilic bacterium Colwellia psychrerythraea that undergoes FMN-induced conformational changes
The ubiX gene of Colwellia psychrerythraea strain 34H encodes a 3-octaprenyl-4-hydroxybenzoate carboxylase (CpsUbiX, UniProtKB code: Q489U8) that is involved in the third step of the ubiquinone biosynthesis pathway and harbors a flavin mononucleotide (FMN) as a potential cofactor. Here, we report the crystal structures of two forms of CpsUbiX: an FMN-bound wild type form and an FMN-unbound V47S...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- Microbiology
دوره 150 Pt 10 شماره
صفحات -
تاریخ انتشار 2004