Zinc in lipase L1 from Geobacillus stearothermophilus L1 and structural implications on thermal stability

• Published in: FEBS letters 2005-06, Vol.579 (16), p.3461-3466
• Main Authors: Choi, Won-Chan, Kim, Myung Hee, Ro, Hyeon-Su, Ryu, Sang Ryeol, Oh, Tae-Kwang, Lee, Jung-Kee
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacillaceae - enzymology; Binding Sites - genetics; Catalytic Domain; circular dichroism; Enzyme Stability; Geobacillus; Geobacillus stearothermophilus; Geobacillus stearothermophilus L1; glutathione S-transferase; GST; HEPES; ICP-AES; inductively coupled plasma-atomic emission spectrometry; Lipase - chemistry; Lipase - genetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; N,N,N′,N′-tetrakis (2-pyridyl-methyl) ethylenediamine; N-(2-hydroxyethyl) piperazine-N′-2-ethanesulfonic acid; Protein Denaturation; Protein Structure, Tertiary; TBN; Temperature; Thermostable lipase; TPEN; tributyrin; Triton X-100; TX100; Zinc - chemistry; Zn 2; Zn2
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/j.febslet.2005.05.016

Lipase L1 from Geobacillus stearothermophilus L1 contains an unusual extra domain, making a tight intramolecular interaction with the main catalytic domain through a Zn 2+-binding coordination. To elucidate the role of the Zn 2+, we disrupted the Zn 2+-binding site by mutating the zinc-ligand residues (H87A, D61A/H87A, and D61A/H81A/H87A/D238A). The activity vs. temperature profiles of the mutant enzymes showed that the disruption of the Zn 2+-binding site resulted in a notable decrease in the optimal temperature for maximal activity from 60 to 45–50 °C. The mutations also abolished the Zn 2+-induced thermal stabilization. The wild-type enzyme revealed a 34.6-fold increase in stabilization with the addition of Zn 2+ at 60 °C, whereas the mutant enzymes exhibited no response to Zn 2+. Additional circular dichroism spectroscopy studies also confirmed the structural stabilizing role of Zn 2+ on lipase L1 at elevated temperatures.