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＜Doctoral Thesis＞
Studies on enzymatic properties and crystal structure of L-leucine dehydrogenase from a psychrophilic bacterium Sporosarcina psychrophila
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| Abstract | L-Leucine dehydrogenase (LeuDH, L-Ieucine: NAD^+ oxidoreductase, deaminating, EC 1.4.1.9) is an oxidoreductase that catalyzes the reversible deamination of L-Ieucine to 4-methyl-2-oxopentanoate in pre...sence of NAD^+ as a cofactor. The enzymes from mesophiles and thermophiles have been so far purified, characterized, and utilized for the L-amino acid determination and production, but not yet from psychrophiles. In this study, LeuDH activity was screened in six psychrophilic bacteria, and the highest levels of enzyme activity were found in Sporosarcina psychrophila DSM 3. The enzyme was purified from S. psychrophila cells to homogeneity and characterized. The protein had an octameric structure with identical 43 ?kDa subunits, giving a total molecular mass of about 340 kDa. The enzyme exhibited the highest activity at 5WC and exhibited one-tenth of that activity even at temperatures as low as 0°C. The enzyme lost no activity with incubation at temperatures lower than 40 °C for 40 min, but did marked loss of the activity higher than 50°C, indicating that the psychrophilic enzyme exhibits less thermostability compared with those of mesophilic and thermophilic LeuDHs. The optimum pHs were 11 for deamination of L-Ieucine and 9 for amination of 4-methyl-2-oxopentanoate. The Km values for L-leucine and NAD^+ at 20°C were 0.65 and 0.015 mM, respectively. Comparison of enzymatic characterizations showed that the catalytic properties of S. psychrophila LeuDH were similar to those of LeuDHs from mesophilic Lysinibacillus sphaericus and thermophuilic Geobacillus stearothermophilus, except for its relatively higher activity even at lower temperature like 10℃. Crystal structural analysis of S. psychrophila LeuDH was determined, showing the first success of 3-dimensional structure for psychrophilic LeuDH. According to the structural comparison of the psychrophilic LeuDH with that of the L. sphaericus one, only minor alterations such as the reduced hydrophobic interactions and number of hydrogen bonds within subunit and at the interfaces between subunits was found, although the total structure was quite similar to each other. The results obtained in this thesis showed that as the first LeuDH isolated from a psychrophilic bacterium, the S. psychrophila enzyme may be useful for practical applications due to its catalytic efficiency even at low temperatures. In addition, the structural analysis of psychrophilic enzyme may afford us someinformation about relationships between the function and structure with respect to higher reactivity of psychrophilic enzymes at low temperatures, although much more detailed biochemical and genetic engineering analyses about the relationships are needed.show more |
| Table of Contents | General introduction Chapter 1 Screening of L-leucine dehydrogenase among psychrophilic bacteria Chapter 2 Purification of L-leucine dehydrogenase from psychrophilic bacterium Sporosarcina psychrophila Chapter 3 Enzymatic Characterization of L-leucine dehydrogenase from psychrophilic bacterium Sporosarcina psychrophila Chapter 4 Gene sequence determination and expression of Sporosarcina psychrophila L-leucine dehydrogenase Chapter 5 Crystallization and structural analysis of Sporosarcina psychrophila L-leucine dehydrogenase |
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| Created Date | 2013.07.10 |
| Modified Date | 2023.12.08 |