Structures of l-asparaginase from Bacillus licheniformis Reveal an Essential Residue for its Substrate Stereoselectivity

Abstract

L-Asparaginase, which catalyzes the hydrolysis of L-asparagine, is an important enzyme in both the clinical and food industry. Exploration of efficient L-asparaginase with high substrate specificity, especially high chiral selectivity, is essential for extending its use. Herein, various crystal structures of type I L-asparaginase from Bacillus licheniformis (BlAsnase) have been resolved, and we found that there are two additional tyrosines in BlAsnase, contributing to the binding and catalysis of D-asparagine. Strikingly, the substitution of Tyr278 with methionine impaired the interaction with D-asparagine via water molecules due to the small hydrophobic side chain of methionine, which forced the ligand to the deep side of the active site toward the catalytic residues and thus resulted in the loss of hydrolyzing function. Our investigation of the substrate recognition mechanism of BlAsnase is significant for both a better understanding of L-asparaginase and its rational design to achieve high specificity for clinical and industrial applications.

Structural and biochemical properties of L‐asparaginase

“…A recent report describes seven structures of BlA, determined at the resolution ranging from 1.8 to 2.25 Å [109]. These results are quite intriguing, as this ASNase appears to combine properties of enzymes from different subclasses.…”

“…Historically, ASNases have been assigned to the type I category when (a) their sequences are more homologous to EcAI than to EcAII, (b) they are cytosolic rather than periplasmic, (c) their affinity for the primary substrate ( l ‐Asn) is relatively low (in the m m range, as reflected by the K M value), and (d) they are tetrameric. Based on these criteria, structures of type I ASNases from three organisms are currently available (structure of the fourth ASNase was recently published, but the coordinates are not yet released [109]). Although dimeric ASNases from archaea are often referred to as type I ASNases, they are considered here as being distinct.…”

Characterization of a Novel L-Asparaginase from Mycobacterium gordonae with Acrylamide Mitigation Potential

“…GmASNase was a tetramer composed of four identical monomers, which could also be said to be a dimer of two intimate dimers. There was a highly flexible "β-hairpin" structure in the N-terminal domain, which is responsible for substrate entry and product release in an open and closed manner [38]. The subunit of GmASNase consists of an N-terminal and a C-terminal domain connected by a linker formed by 173-200 residues (Figure 2C).…”

“…GmASNase exhibited strict specificity towards L-asparagine (100%), but low activity on D-asparagine (22%) (Figure 5A). Ran et al found that the high catalytic activity of D-asparaginase might be associated with the particularity of the substrate binding pocket by multipoint mutation and structure parsing [38]. Moreover, GmASNase displayed a Km value of 6.025 mM, which is lower than those of many other L-asparaginases from different species (Table S1), such as Rhizobium etli (8.9 mM), P. furiosus (12 mM), T. gammatolerans EJ3 (10 mM), and P. oyzihabitans (10.0 mM).…”

Selective Immobilization of His‐Tagged Phosphomannose Isomerase on Ni Chelated Nanoparticles with Good Reusability and Activity

“…Enzymes are very significant biocatalysts, which are widely used in food industry, [1] biocatalysis, [2] leather industry, [3] biosensor development, [4] biodiesel production [5] and other application fields owing to their high substrate selectivity, high catalytic efficiency, and mild reaction conditions [6–8] . However, there are some disadvantages in enzyme preparation, such as poor stability, [9] strict storage and transportation conditions, and poor recyclability, which greatly limits their practical applications.…”