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.

Enzymatic features that determine transglycosylating activity have been investigated through site-directed mutagenesis studies on two family 18 chitinases, ChiA and ChiB from Serratia marcescens, with inherently little transglycosylation activity. The activity was monitored for the natural substrate (GlcNAc)(4) using mass spectrometry and HPLC. Mutation of the middle Asp in the diagnostic DxDxE motif, which interacts with the catalytic Glu during the catalytic cycle, yielded the strongly transglycosylating mutants ChiA-D313N and ChiB-D142N, respectively. Mutation of the same Asp(313/142) to Ala or the mutation of Asp(311/140) to either Asn or Ala had no or much smaller effects on transglycosylating activity. Mutation of Phe(396) in the +2 subsite of ChiA-D313N to Trp led to a severalfold increase in transglycosylation rate while replacement of aromatic residues with Ala in the aglycon (sugar acceptor-binding) subsites of ChiA-D313N and ChiB-D142N led to a clear reduction in transglycosylating activity. Taken together, these results show that the transglycosylation properties of family 18 chitinases may be manipulated by mutations that affect the configuration of the catalytic machinery and the affinity for sugar acceptors. The hypertransglycosylating mutant ChiA-D313N-F396W may find applications for synthetic purposes.

a b s t r a c tThe contributions of the -3 subsite and a putative +3 subsite to substrate positioning in ChiA from Serratia marcescens have been investigated by comparing how ChiA and its -3 subsite mutant W167A interact with soluble substrates. The data show that Trp -GlcNAc stacking in the -3 subsite rigidifies the protein backbone supporting the formation of the intermolecular interaction network that is necessary for the recognition and positioning of the N-acetyl groups before the -1 subsite. The +3 subsite exhibits considerable substrate affinity that may promote endo-activity in ChiA and/or assist in expelling dimeric products from the +1 and +2 subsites during processive hydrolysis.

Enzymatic depolymerization of polysaccharides is a key technology in the biorefining of biomass. The enzymatic conversion of the abundant insoluble polysaccharides cellulose and chitin is of particular interest and complexity, because of the bi-phasic nature of the process, the seemingly complicated tasks faced by the enzymes, and the importance of these conversions for the future biorefinery. Here we review recent work on family 18 chitinases that sheds light on important aspects of the catalytic action of these depolymerizing enzymes, including the structural basis of processivity and its direction, the energies involved in substrate-binding and displacement.

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