Informational Suppression to Investigate Structural Functional and Evolutionary Aspects of the Erwinia chrysanthemiCellulase EGZ

Abstract

The cellulase EGZ produced by the plant pathogen Erwinia cyrysaqnthemibelongs to family 5 of the β-glycohydrolases (also referred to as cellulase family A), which contains over 40 members from Gram-negative and Gram-positive bacteria and fungi. Amber mutations were introduced into 16 codons of the celZgene encoding EGZ. Targeted residues include: (1) two Glu, two His and one Arg residue, strictly conserved throughout family 5; (2) one Arg and one His residue conserved in sub-family 5-2; and (3) one His and six Arg residues not conserved at all. Each amber allele was introduced into 13 Escherichia colistrains each carrying a different suppressor tRNA that inserts an amino acid at the mutual position. In vivostability of the mutated forms of EGZ and their cellulase activity were analysed as well as suppression efficiency. For some positions of particular interest, missense mutations were introduced into the celZgene either to confirm the effect of the suppressor-mediated amino acid substitution or to broaden the spectrum of mutations available. The substitution patterns of the two Glu positions were interpretable in the light of the stereospecificity of the reaction catalysed by EGZ: Glu133 and Glu220 are proposed to act as a proton donor and as a nucleophile, respectively, forming the glycosyl-enzyme intermediate. Substitution at His-occupied positions, including two non-conserved positions, yielded proteins affected in their catalytic activity but not their in vivostability. In particular, evidence was obtained for His at position 98 to be involved in interactions with the substrate. The view that Arg residues are important in stabiliizing proteins was supported by the identification of three Arg residues, whose substitution yielded thermosensitive forms of EGZ. In addition, Pro substitutions of any of the six Arg residues altered protein stability in vivobut the substitutions scored almost neutral for activity. Five positions, predicted to be within α-helices, were found to be susceptible to Pro substitutions (but not to Ala) with respect to stability in vivo. Overall, the systematic alteration of all His and Arg residues coupled with the simultaneous analysis of activity and in vivostability allowed us to demonstrate that substitution matrices vary at each position and for each biological property considered. Ideally, therefore, substitution matrices used in sequence alignment procedures should be reconsidered as position-specific and as property-specific. Allowed amino acid substitutions at conserved and non-conserved positions were compared with natural variability occurring in the set of 40 cellulases present in family 5, thereby providing us with a direct comparison between laboratory-induced and naturally occurring evolution and between experimentally and theoretically based substitution matrices.