Crystal structures of the elusive Rhizobium etli l-asparaginase reveal a peculiar active site

Abstract

Rhizobium etli, a nitrogen-fixing bacterial symbiont of legume plants, encodes an essential l-asparaginase (ReAV) with no sequence homology to known enzymes with this activity. High-resolution crystal structures of ReAV show indeed a structurally distinct, dimeric enzyme, with some resemblance to glutaminases and β-lactamases. However, ReAV has no glutaminase or lactamase activity, and at pH 9 its allosteric asparaginase activity is relatively high, with Km for l-Asn at 4.2 mM and kcat of 438 s−1. The active site of ReAV, deduced from structural comparisons and confirmed by mutagenesis experiments, contains a highly specific Zn2+ binding site without a catalytic role. The extensive active site includes residues with unusual chemical properties. There are two Ser-Lys tandems, all connected through a network of H-bonds to the Zn center, and three tightly bound water molecules near Ser48, which clearly indicate the catalytic nucleophile.

Structural and biophysical studies of new L-asparaginase variants: lessons from random mutagenesis of the prototypic Escherichia coli Ntn-amidohydrolase

“…S3 and S4); thus, the reasons for aborted autoprocessing are probably related to incorrect positioning of the linker and scissile bond or an incorrect arrangement of water molecules or of the oxyanion hole. As the exact role of the intrinsically disordered linker is not clear at present (Loch & Jaskolski, 2021), its impact on autoprocessing should be investigated in detail in future experiments. To date, there are no crystallographic data that allow a comprehensive analysis of the conformation of the linker: even in the structures of the uncleaved EcAIII mutant T179A (PDB entry 3zal) or of uncleaved HsAIII (PDB entry 4osx) a large portion of the linker could not be traced in the electron-density maps (Michalska et al, 2008;Su et al, 2013).…”

“…l-Asparaginases hydrolyze l-asparagine to l-aspartic acid and ammonia. Based on their amino-acid sequences and the architecture of the protein fold, l-asparaginases are divided into three classes (Loch & Jaskolski, 2021;da Silva et al, 2022): class 1 (bacterial-type), class 2 (plant-type) and class 3 (Rhizobium etli-type) (Loch et al, 2021). The prototypic members of class 1 include the Escherichia coli enzyme EcAII and its homolog from Erwinia chrysanthemi 1 (ErAII), which are used to treat acute lymphoblastic leukemia (ALL; Sugimoto et al, 2015).…”

Massive annotation of bacterial l-asparaginases reveals their puzzling distribution and frequent gene transfer events

“…the tightly bound zinc cation, with its open coordination sphere (complemented by a water molecule), is not required for the l-asparaginase activity of ReAV 10 . l-Asparaginases from different Classes show distinct architecture and oligomeric states.…”

“…ReAV is a homodimer of two subunits, each built of two domains: the catalytic and dimer stabilization domain. In contrast to Class 1 and Class 2 enzymes, ReAV binds a zinc cation in an unusual 2xCys/Lys/water coordination pattern 10 .…”

“…S1d,e), but more similar to serine β-lactamases or penicillin binding proteins (PBPs). The only known structure of a Class 3 enzyme has been recently published 10 for the archetypical ReAV protein from R. etli. ReAV is a homodimer of two subunits, each built of two domains: the catalytic and dimer stabilization domain.…”