A psychrophilic leucine dehydrogenase from Sporosarcina psychrophila: Purification, characterization, gene sequencing and crystal structure analysis

Ying Zhao, Taisuke Wakamatsu, Katsumi Doi, Haruhiko Sakuraba, Toshihisa Ohshima

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17 Citations (Scopus)

Abstract

Leucine dehydrogenase (LeuDH, l-leucine: NAD + oxidoreductase, deaminating, EC 1.4.1.9) was screened in six psychrophilic bacteria, and the highest levels of enzyme activity were found in Sporosarcina psychrophila DSM 3. As the first LeuDH from a psychrophilic bacterium, the enzyme was purified 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 50 °C 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 there was marked loss of activity with incubations at temperatures higher than 50 °C. The optimum pHs were 11 for deamination of l-leucine and 9 for amination of 4-methyl-2-oxopentanoate. The K m values for l-leucine and NAD + at 20 °C were 0.65 and 0.015 mM, respectively. The catalytic properties of S. psychrophila LeuDH were similar to those of LeuDHs from Lysinibacillus sphaericus and Geobacillus stearothermophilus, except for its lower optimal reaction temperature and thermostability at low temperatures. Crystal structural analysis of S. psychrophila LeuDH showed the total structure to be similar to that of the L. sphaericus enzyme, except minor alterations reduced the hydrophobic interactions and hydrogen bonds within and between subunits.

Original languageEnglish
Pages (from-to)65-72
Number of pages8
JournalJournal of Molecular Catalysis B: Enzymatic
Volume83
DOIs
Publication statusPublished - Nov 1 2012

All Science Journal Classification (ASJC) codes

  • Catalysis
  • Bioengineering
  • Biochemistry
  • Process Chemistry and Technology

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