The Non-Catalytic Amino Acid Asp₄₄₆ Is Essential for Enzyme Activity of the Modular Endocellulase Cel9 from Myxobacter sp. AL-1

Abstract

The modular endocellulase Cel9 of the bicistronic operon cel9-cel48 of Myxobacter sp. AL-1 shares not only amino acid sequence similarity but also biochemical properties similar to those of Thermobifida fusca endo/exocellulase E4. Amino acid alignments of a T. fusca E4 cellulase subfamily of family 9 cellulases revealed that Asp₄₄₆ of Myxobacter sp. AL-1 Cel9, a putatively noncatalytic residue, is highly conserved in one of the catalytic domains of this subfamily. Directed mutagenesis of residue aspartate (Asp₄₄₆) to alanine generated a Cel9 mutant that lost more than 99% of its activity, suggesting that Asp₄₄₆ plays an essential structural role in Cel9 during cellulose degradation. Owing to its high degree of conservation and essential role, we propose that Asp₄₄₆ of Myxobacter sp. AL-1 Cel9 is a good landmark that distinguishes members of the E4 subfamily of family 9 cellulases.