Rhizobium etli has two L-asparaginases with low sequence identity but similar structure and catalytic center

Abstract

The genome of Rhizobium etli, a nitrogen-fixing bacterial symbiont of legume plants, encodes two L-asparaginases, ReAIV and ReAV, that have no similarity to the well characterized enzymes of class 1 (bacterial type) and class 2 (plant type). It has been hypothesized that ReAIV and ReAV might belong to the same structural class 3 despite their low level of sequence identity. When the crystal structure of the inducible and thermolabile protein ReAV was solved, this hypothesis gained a stronger footing because the key residues of ReAV are also present in the sequence of the constitutive and thermostable ReAIV protein. High-resolution crystal structures of ReAIV now confirm that it is a class 3 L-asparaginase that is structurally similar to ReAV but with important differences. The most striking differences concern the peculiar hydration patterns of the two proteins, the presence of three internal cavities in ReAIV and the behavior of the zinc-binding site. ReAIV has a high pH optimum (9–11) and a substrate affinity of ∼1.3 mM at pH 9.0. These parameters are not suitable for the direct application of ReAIV as an antileukemic drug, although its thermal stability and lack of glutaminase activity would be of considerable advantage. The five crystal structures of ReAIV presented in this work allow a possible enzymatic scenario to be postulated in which the zinc ion coordinated in the active site is a dispensable element. The catalytic nucleophile seems to be Ser47, which is part of two Ser–Lys tandems in the active site. The structures of ReAIV presented here may provide a basis for future enzyme-engineering experiments to improve the kinetic parameters for medicinal applications.

Biochemical characterization of L-asparaginase isoforms from Rhizobium etli—the boosting effect of zinc

“…It is prominently represented by two isoforms, ReAIV and ReAV, derived from Rhizobium etli , a nitrogen-fixing bacterial symbiont of the legume common bean. ReAIV is constitutive and thermostable, while ReAV is inducible and thermolabile, the denaturation temperatures being, respectively, 72°C and 51°C ( Loch et al, 2023 ). The level of sequence homology between ReAIV and ReAV is rather low, with ∼31% identity and ∼45% similarity, and both proteins have no sequence similarity to other known enzymes with this activity.…”

“…The level of sequence homology between ReAIV and ReAV is rather low, with ∼31% identity and ∼45% similarity, and both proteins have no sequence similarity to other known enzymes with this activity. Despite their sequence differences, the two isoforms show high structural similarity, with the caveat that the longer ReAV sequence (367 vs. 335 residues) has additional structural elements (such as longer loops) that make it a less compact protein ( Loch et al, 2023 ) ( Figure 1A ). The crystal structures of both enzymes revealed that their unusual active site contains a highly specific Zn 2+ binding site, formed by a Lys and two Cys residues ( Figure 1B ), adjacent to two conspicuous Ser-Lys tandems ( Loch et al, 2021 ; Loch et al, 2023 ).…”

“…Despite their sequence differences, the two isoforms show high structural similarity, with the caveat that the longer ReAV sequence (367 vs. 335 residues) has additional structural elements (such as longer loops) that make it a less compact protein ( Loch et al, 2023 ) ( Figure 1A ). The crystal structures of both enzymes revealed that their unusual active site contains a highly specific Zn 2+ binding site, formed by a Lys and two Cys residues ( Figure 1B ), adjacent to two conspicuous Ser-Lys tandems ( Loch et al, 2021 ; Loch et al, 2023 ). The ReAV and ReAIV enzymes are dimeric but, unlike in Class 1 and 2, the catalytic center is structurally composed of residues from one subunit only.…”

Probing the active site of Class 3 L-asparaginase by mutagenesis. I. Tinkering with the zinc coordination site of ReAV

“…In order to find the optimal zinc concentration for WT ReAV and its mutants, L-asparaginase activity was determined by the Nessler method using the protocol from Loch et al (2023) with 10 mM L-Asn in 20 mM Tris-HCl buffer pH 9.0 and enzyme concentration in the range of 60 nM–3.8 µM, in the presence of zinc ions in concentration varied from 1 to 100 μM (as was the case of the WT and K138A proteins), and additionally extended to the nanomolar range (150–500 nM) in the case of the H139A mutant. The relative activity expressed as the percentage of enzyme activity without zinc supplementation was calculated and plotted to compare the shape of the zinc profiles and the level of response to Zn 2+ .…”

“…The sequences of these two proteins show a low level of identity (∼30%) and both are significantly different from the sequences of Class 1 and Class 2 asparaginases ( Loch and Jaskolski, 2021 ). The recently reported crystal structure of ReAIV ( Loch et al, 2023 ) showed that despite the low sequence identity, the two enzymes have basically the same 3D structure.…”