Structural characterization of a soil viral auxiliary metabolic gene encoding a functional chitosanase

Abstract Metagenomics is unearthing the previously hidden world of soil viruses1,2. Many soil viral sequences in metagenomes contain putative auxiliary metabolic genes (AMGs) that are not associated with viral replication. To date only one soil viral AMG has been expressed3 and none has a solved structure. Here, we aimed to establish that AMGs on soil viruses actually produce functional, active proteins. We focused on AMGs that potentially encode chitosanase enzymes that metabolize chitin – a common carbon polymer. Glycoside hydrolase family 75 viral chitosanase genes were identified from environmental metagenomes. Several of these were expressed and functionally screened. One expressed protein showing endo-chitosanase activity (V-Csn) was crystalized and structurally characterized at ultra-high resolution, thus representing the first structure of a soil viral AMG product. A structure for an inactive mutant with a bound substrate (chitosan oligomer) was also determined. Conserved active site residues in V-Csn resided in a cleft between two domains. These structures provided details about the active site, and together with structure models determined using AlphaFold2, facilitated understanding of substrate specificity and enzyme mechanism. These findings support the hypothesis that soil viruses contribute auxiliary functions to their hosts. Soil viral chitosanase AMG products that assist chitin decomposition may therefore play a previously unrecognized essential role in soil carbon cycling.