甲烷太古生物之相容質Nε-acetyl-β-lysine自體生合成基因以及酵素特性分析

Abstract

To adapt to the broad range of salt concentrations, methanogenic archaea accumulate potassium, α-glutamate, glycine betaine, β-glutamate, β-gultamine and Ne-acetyl-β-lysine as compatible solutes (osmolytes) to encounter the osmotic stress. The accumulation of Ne-acetyl-β-lysine as osmolyte is ubiquitous among methanogenic archaea, but not for other organisms. Ne-acetyl-β-lysine is synthesized from lysine through lysine 2,3-aminomutase (AblA) to form β-lysine and the acetyl group is further transferred to β-lysine by β-lysine acetyltransferase (AblB). The ablA and ablB genes were screened and obtained through PCR and Southern hybridization techniques from the marine Methanosarcina mazei N2M9705, halotolerant Methanocalculus chunghsingensis K1F9705bT and halophilic Methanohalophilus portucalensis FDF1T. The amino acid sequences of lysine 2,3-aminomutase (AblA) from these three methanogenic archaea contains ligands of the Fe/S cluster, SAM binding domain, PLP binding site and zinc binding sites; and β-lysine acetyltransferase (AblB) is a member of the GNAT [GCN5 (general control non-derepressible 5)-related N-acetyltransferase] superfamily of enzymes that contain R/Q-G/K-K/L-G-H/L-M/S-K/G segment. Compared the phylogenetic relationships of ablA and ablB genes along with 16S RNA genes, suggested the possible horizontal gene transfer may occur within halotolerant, halophilic methanogen and halophilic green-sulfur bacteria; or within Methanosarcina sp. and Bacillus/ Clostridium. Northern hybridization results showed that ablA and ablB were in one transcribed unit, and the osmolyte Ne-acetyl-β-lysine biosynthetic related genes from M. portucalensis FDF1T were salt up-regulated, but not immediately response to the temperature stress. The recombinant MpAblB and McAblB showed the high binding affinity to acetyl-CoA with Km value at 54.348 μM and 58.140 μM, respectively. Whereas the binding affinity to α-lysine were low with K0.5 value at 40~310 mM, suggested the AblB exihibits cooperative binding of α-lysine. Low affinity to α-lysine may due to the high specificity to β-lysine of the β-lysine acetyltransferase. The acetyltransferase activity of the recombinant MpAblB and McAblB were repressed by increasing level of potassium or sodium ions. Extremozymes from halotolerant and halophilic archaea were capable of salt-tolerant, solven-tolerant and retain catalytic activity in environments with low water activity. Lysine 2,3-aminomutase from methanoarchaea, especially from the halophilic methanogens, could act as potential biocatalysts for the synthesis of β-lysine, which can be applied in pharmaceutical applications.