Fig. 2. The activity and structure of the soil viral chitosanase enzyme V-Csn.

a Activity of V-Csn was assayed by monitoring azurine absorbance at 590 nm following its release as soluble azurine-sugar fragments from an insoluble azurine cross-linked chitosan substrate (AZCL-chitosan). Reactions were performed at room temperature over a range of pH values in acetate buffer. The data represent the mean of replicate experiments (n = 3) with the standard deviation indicated by the error bars. Source data are provided as a Source Data file. b Partial sequence alignment of V-Csn with three chitosanase enzymes from Aspergillus fumigatus, Fusarium solani and Streptomyces avermitilis. The secondary structure for V-Csn is indicated above the alignment. Four conserved acidic residues potentially involved in catalysis are colored blue and red. Alignment of available sequences of GH75 family enzymes show that only six positions are universally conserved, these four acidic residues and two glycine residues, as indicated by the asterisks beneath the alignment. c Release of azurine by wild-type V-Csn and two constructs containing either a D148N or E157Q substitution, in acetate buffer at pH 5.1. The data represent the mean of replicate experiments (n = 3) with the standard deviation indicated by the error bars. Source data are provided as a Source Data file. d Ribbon representation of the apo1 form of the V-Csn enzyme with the two structural domains colored light green (Domain-1) and pink (Domain-2). The secondary structure nomenclature is given, along with the locations of the N- and C-termini. e Superposition of a dimer constructed from V-Csn apo1 (molecule 1, yellow ribbon) and a crystallographic symmetry partner (molecule 2, blue ribbon), and the V-Csn apo2 homodimer (gray ribbons). The secondary structure elements which comprise the dimer interface are indicated. Secondary structure elements labeled with a prime represent the symmetry related apo1 molecule. f The V-Csn apo2 dimer showing molecule 1 as a yellow molecular surface and molecule 2 as a blue ribbon. The contact area between the two molecules is shown in light blue on the yellow molecule 1 surface. Source data are available as a Source Data file.