Structural Superimposition

Abstact

Fungal class 3 L-asparaginases were identified based on sequence similarity to the bacterial enzyme ReAV from Rhizobium etli. Here, we present an integrative functional and structural characterization of six class 3 L-asparaginases derived from pathogenic fungi. Kinetic analyses revealed distinct catalytic profiles, including differences in substrate affinity and turnover rates. For three enzymes, a total of six high-resolution crystal structures were determined, capturing multiple active site states. Structural comparisons confirm the conserved fold characteristic of class 3 L-asparaginases and reveal unexpected modifications at the catalytic Ser51 nucleophile. In all three structurally characterized enzymes, Ser51 is esterified by orthoborate, which bears a secondary esterification by glycerol or ethylene glycol, as observed under the crystallization conditions used in this study. The physiological relevance of this modification remains unknown, but it provides new insight into the chemical reactivity of the active site and suggest a previously unrecognized mechanism. To explore the susceptibility of fungal L-asparaginases to small-molecule inhibition, we performed docking-based virtual screening using an atomic resolution (~1 A) crystal structure of the Botryosphaeria parva enzyme (BpA). Screening of over two million compounds yielded candidate molecules predicted to bind within the catalytic pocket. Experimental evaluation of five compounds identified one preliminary hit, L0911, displaying measurable inhibition of BpA, with IC(50) of ~800 muM. Together, these results expand our understanding of class 3 L-asparaginase structure and function, reveal unexpected active-site chemistry, and provide a starting point for the development of small-molecule inhibitors targeting the fungal enzymes as potential fungicides.