8bi3
From Proteopedia
Structure of E. coli Class 2 L-asparaginase EcAIII, mutant M200W (crystal M200W#1)
Structural highlights
FunctionIAAA_ECOLI Degrades proteins damaged by L-isoaspartyl residue formation (also known as beta-Asp residues). Degrades L-isoaspartyl-containing di- and maybe also tripeptides. Also has L-asparaginase activity, although this may not be its principal function.[1] May be involved in glutathione, and possibly other peptide, transport, although these results could also be due to polar effects of disruption.[2] Publication Abstract from PubMedThe 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. This article is protected by copyright. All rights reserved. The effects of nature-inspired amino acid substitutions on structural and biochemical properties of the E. coli L-asparaginase EcAIII.,Janicki M, Sciuk A, Zielezinski A, Ruszkowski M, Ludwikow A, Karlowski WM, Jaskolski M, Loch JI Protein Sci. 2023 Apr 24:e4647. doi: 10.1002/pro.4647. PMID:37095066[3] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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