The effects of nature-inspired amino acid substitutions on structural and biochemical properties of the E. coli L-asparaginase EcAIII.

Autor: Janicki M; Department of Biotechnology, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznań, Poland., Ściuk A; Department of Crystal Chemistry and Crystal Physics, Faculty of Chemistry, Jagiellonian University, Krakow, Poland., Zielezinski A; Department of Computational Biology, Faculty of Biology, Adam Mickiewicz University, Poznań, Poland., Ruszkowski M; Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland., Ludwików A; Department of Biotechnology, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznań, Poland., Karlowski WM; Department of Computational Biology, Faculty of Biology, Adam Mickiewicz University, Poznań, Poland., Jaskolski M; Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland.; Department of Crystallography, Faculty of Chemistry, Adam Mickiewicz University, Poznań, Poland., Loch JI; Department of Crystal Chemistry and Crystal Physics, Faculty of Chemistry, Jagiellonian University, Krakow, Poland.
Jazyk: angličtina
Zdroj: Protein science : a publication of the Protein Society [Protein Sci] 2023 Jun; Vol. 32 (6), pp. e4647.
DOI: 10.1002/pro.4647
Abstrakt: The Escherichia coli enzyme EcAIII catalyzes the hydrolysis of L-Asn to L-Asp and ammonia. Using a nature-inspired mutagenesis approach, we designed and produced five new EcAIII variants (M200I, M200L, M200K, M200T, M200W). The modified proteins were characterized by spectroscopic and crystallographic methods. All new variants were enzymatically active, confirming that the applied mutagenesis procedure has been successful. The determined crystal structures revealed new conformational states of the EcAIII molecule carrying the M200W mutation and allowed a high-resolution observation of an acyl-enzyme intermediate with the M200L mutant. In addition, we performed structure prediction, substrate docking, and molecular dynamics simulations for 25 selected bacterial orthologs of EcAIII, to gain insights into how mutations at the M200 residue affect the active site and substrate binding mode. This comprehensive strategy, including both experimental and computational methods, can be used to guide further enzyme engineering and can be applied to the study of other proteins of medicinal or biotechnological importance.
(© 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)
Databáze: MEDLINE
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