Klebsiella phage KP34gp57 capsular depolymerase structure and function: from a serendipitous finding to the design of active mini-enzymes against K. pneumoniae

Abstract

ABSTRACT Virion-associated depolymerases are large trimeric and multi-domain proteins that constitute the phage arsenal to degrade the polysaccharide layers in their bacterial host. Thus, as recombinant proteins, they are endowed with huge potential in biotechnology and medicine. In this study, we elucidated the structural and functional features of the capsular depolymerase KP34gp57 from the Klebsiella phage KP34. Based on the crystal structure and site-directed mutagenesis, we localized the key catalytic residues in an intra-subunit deep groove. Moreover, we engineered several N- and C-terminally truncated versions of KP34gp57 to dissect the role of each domain in the enzyme’s stability and catalytic activity. Serendipitously, our studies revealed C-terminally trimmed KP34gp57 variants that did not trimerize and were sufficiently stable to preserve full catalytic activity as monomers. The elaboration of trimmed monomeric and fully active phage depolymerases is innovative in the field, as no previous example exists apart from bacterial enzymes. Mini phage depolymerases can be optionally combined within chimeric enzymes to extend their activity range, facilitating their use in stand-alone treatments. Moreover, the intra-subunit and inter-subunit locations of the catalytic pocket in phage depolymerases might suggest differences in their evolutionary origin. IMPORTANCE In this work, we determined the structure of Klebsiella phage KP34p57 capsular depolymerase and dissected the role of individual domains in trimerization and functional activity. The crystal structure serendipitously revealed that the enzyme can exist in a monomeric state once deprived of its C-terminal domain. Based on the crystal structure and site-directed mutagenesis, we localized the key catalytic residues in an intra-subunit deep groove. Consistently, we show that C-terminally trimmed KP34p57 variants are monomeric, stable, and fully active. The elaboration of monomeric, fully active phage depolymerases is innovative in the field, as no previous example exists. Indeed, mini phage depolymerases can be combined in chimeric enzymes to extend their activity ranges, allowing their use against multiple serotypes.