Journal Article

PUBDB-2025-02290

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Probing the Active Site of Class 3 L-Asparaginase by Mutagenesis: Mutations of the Ser-Lys Tandems of ReAV

Pokrywka, K. (Corresponding author)Extern*EMBL* ; Grzechowiak, M. ; Sliwiak, J. ; Worsztynowicz, P. ; Loch, J. I. ; Ruszkowski, M. ; Gilski, M. ; Jaskolski, M. (Corresponding author)

2025
MDPI Basel

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Please use a persistent id in citations: doi:10.3390/biom15070944  doi:10.3204/PUBDB-2025-02290

Abstract: The ReAV enzyme from Rhizobium etli, a representative of Class 3 L-asparaginases, is sequentially and structurally different from other known L-asparaginases. This distinctiveness makes ReAV a candidate for novel antileukemic therapies. ReAV is a homodimeric protein, with each subunit containing a highly specific zinc-binding site created by two cysteines, a lysine, and a water molecule. Two Ser-Lys tandems (Ser48-Lys51, Ser80-Lys263) are located in the close proximity of the metal binding site, with Ser48 hypothesized to be the catalytic nucleophile. To further investigate the catalytic process of ReAV, site-directed mutagenesis was employed to introduce alanine substitutions at residues from the Ser-Lys tandems and at Arg47, located near the Ser48-Lys51 tandem. These mutational studies, along with enzymatic assays and X-ray structure determinations, demonstrated that substitution of each of these highly conserved residues abolished the catalytic activity, confirming their essential role in enzyme mechanism.

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Contributing Institute(s):

1. EMBL-User (EMBL-User)

Research Program(s):

1. 6G3 - PETRA III (DESY) (POF4-6G3) (POF4-6G3)

Experiment(s):

1. PETRA Beamline P13 (PETRA III)

Appears in the scientific report 2025

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