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L-Lysine α-oxidase (LysOX) from Trichoderma viride is a homodimeric flavoenzyme that catalyzes the oxidative deamination of L-Lysine to produce α-keto-ε-aminocaproate with ammonia and hydrogen per-oxide. LysOX inhibited the growth of cancer cells but showed relatively low toxicity for normal cells. The full-length cDNA consists of 2,119 bp, and encodes a long N-terminal propeptide composed of 77 resi-dues (Met1-Arg77) and the mature protein (Ala78-Ile617). The LysOX gene was heterologously expressed in Streptomyces lividans TK24 or Escherichia coli SoluBL21. The enzymatic properties of the purified recombinant LysOX, such as substrate specificity, kinetic parameters and thermal stability, are the same as those of the native LysOX. The LysOX precursor (prLysOX) expressed in E. coli shows weak enzymatic activity and is activated by proteolytic processing. The crystal structure of prLysOX revealed that the propeptide of prLysOX indirectly changes the active site structure to inhibit enzyme activity. Moreover, the crystal structures of LysOX and its L-Lysine complex revealed that the hydrogen bonding network formed by Asp212, Asp315 and Ala440 with two water molecules is responsible for the recogni-tion of the ε-amino group of L-Lysine. In addition, a narrow substrate-binding site and acidic surface at the active site entrance both contribute to strict substrate specificity. Mutational analysis demonstrated that Asp212 and Asp315 are essential for substrate recognition, and the D212A/D315A LysOX prefers aromatic amino acids. Furthermore, the structural basis of the substrate specificity change has also been revealed by the structural analysis of the D212A/D315A LysOX and its substrate complexes. |
