Aromatic Residues in the Catalytic Center of Chitinase A from Serratia marcescens Affect Processivity, Enzyme Activity, and Biomass Converting Efficiency

Abstract

The processive Serratia marcescens chitinases A (ChiA) and B (ChiB) are thought to degrade chitin in the opposite directions. A recent study of ChiB suggested that processivity is governed by aromatic residues in the ؉1 and ؉2 (aglycon) subsites close to the catalytic center. To further investigate the roles of aromatic residues in processivity and to gain insight into the structural basis of directionality, we have mutated Trp 167 , Trp 275 , and Phe 396 in the ؊3, ؉1, and ؉2 subsites of ChiA, respectively, and characterized the hydrolytic activities of the mutants toward ␤-chitin and the soluble chitin-derivative chitosan. Although the W275A and F396A mutants showed only modest reductions in processivity, it was almost abolished by the W167A mutation. Thus, although aglycon subsites seem to steer processivity in ChiB, a glycon (؊3) subsite seems to be adapted to do so in ChiA, in line with the notion that the two enzymes have different directionalities. Remarkably, whereas all three single mutants and the W167A/W275A double mutant showed reduced efficiency toward chitin, they showed up to 20-fold higher activities toward chitosan. These results show that the processive mechanism is essential for an efficient conversion of crystalline substrates but comes at a large cost in terms of intrinsic enzyme speed. This needs to be taken into account when devising enzymatic strategies for biomass turnover.

Substrate positioning in chitinase A, a processive chito-biohydrolase fromSerratia marcescens

“…The results also suggest that Trp 167 is important for recruiting substrates to the catalytic center, in line with what has been suggested for a similar residue (Trp 48 ) in the family 18 chitinase Chit-42 of Trichoderma harzianum [34]. Table 1 Sequence determination of CHOS of DP = 5 obtained after hydrolysis of chitosan (F A 0.66) with ChiA-WT and ChiA-W167A to a maximum degree of scission [11]. …”

“…ChiA-W167A and ChiA wildtype (WT) from S. marcescens were over-expressed and purified as described elsewhere [11].…”

“…As a final check of the role of Trp167 in substrate binding and positioning, we analyzed products obtained after degrading a chitosan with a degree of acetylation of 66% with ChiA-WT or ChiA-W167A, to a maximum degree of scission [11]. An impression of product sequences was obtained by sequencing the DP5 product fraction using reducing end labeling and MALDI-TOF-TOF-MS/MS analysis [26].…”

“…In order to bind to and guide the insoluble substrate into the active site cleft, many chitinases, including all those that are known to act processively, have a path of conserved solvent exposed aromatic residues leading from the chitin binding domain to the active site cleft [8][9][10][11][12] (Fig. 1).…”

“…Recently, it was shown that Trp 167 in subsite -3 ( Fig. 1), interacting with the polymeric and ''sliding'' part of the substrate, is of particular importance for the processive action for ChiA [11].…”