Key Residues Affecting Transglycosylation Activity in Family 18 Chitinases: Insights into Donor and Acceptor Subsites

Abstract

Understanding features that determine transglycosylation (TG) activity in glycoside hydrolases is important because it would allow the construction of enzymes that can catalyze controlled synthesis of oligosaccharides. To increase TG activity in two family 18 chitinases, chitinase D from Serratia proteamaculans ( SpChiD) and chitinase A from Serratia marcescens ( SmChiA), we have mutated residues important for stabilizing the reaction intermediate and substrate binding in both donor and acceptor sites. To help mutant design, the crystal structure of the inactive SpChiD-E153Q mutant in complex with chitobiose was determined. We identified three mutations with a beneficial effect on TG activity: Y28A (affecting the -1 subsite and the intermediate), Y222A (affecting the intermediate), and Y226W (affecting the +2 subsite). Furthermore, exchange of D151, the middle residue in the catalytically important DXDXE motif, to asparagine reduced hydrolytic activity ≤99% with a concomitant increase in apparent TG activity. The combination of mutations yielded even higher degrees of TG activity. Reactions with the best mutant, SpChiD-D151N/Y226W/Y222A, led to rapid accumulation of high levels of TG products that remained stable over time. Importantly, the introduction of analogous mutations at the same positions in SmChiA (Y163A equal to Y28A and Y390F similar to Y222A) had similar effects on TG efficiency. Thus, the combination of the decreasing hydrolytic power, subsite affinity, and stability of intermediate states provides a powerful, general strategy for creating hypertransglycosylating mutants of retaining glycoside hydrolases.

Single-molecule imaging analysis reveals the mechanism of a high-catalytic-activity mutant of chitinase A from Serratia marcescens

“…This F396A mutation increased the K d value 2-fold and slightly reduced the processivity compared with WT (11,43). The slightly decreased K m was also observed when Phe-396 was mutated to Trp with D313N (44,45). Taken together, F232W/F396W mutation in SmChiA would be better in guiding the chitin chain, whereby the binding affinity was increased and the degree of processivity was improved as a result of the larger aromatic surface area of Trp compared with Phe.…”

Structural inspection and protein motions modelling of a fungal glycoside hydrolase family 18 chitinase by crystallography depicts a dynamic enzymatic mechanism

“…Previous results reported that the mutation F396A, the equivalent residue in SmChitA, had little impact on hydrolysis of crystalline chitin and only moderate impact on processive ability on chitosan [15] , which was explained as this Phe having little role in ligand binding by calculating the free energy through experimental and MD simulation techniques [42] . However, the replacement of Phe by Trp increases markedly its transglycosilation ability [18] revealing a role in acceptor substrate binding. Nevertheless, it is worth noting that, while loop β6-α6 of the SmChitA is quite conserved with Chit42, the adjacent CID domain is the most variable region and, in particular, SmChitA presents longer β7aβ7b and β7b-β7c loops ( Figure S1 ).…”

“…Although this reaction is secondary to the hydrolytic activity, the balance can be kinetically controlled and has been proved to be modulated by mutagenesis of key residues at the catalytic site, and also by removal of some aromatics at the active site groove of chitinases from Serratia [17] . Thus, mutations leading to decreased rate of hydrolysis and enhanced sugar acceptor-binding access may result in an increased level of TG products [18] . Therefore, a full understanding of the catalytic mechanism and depiction of subsites affinities is required to fully exploit the synthetic use of chitinases.…”

Probing the determinants of the transglycosylation/hydrolysis partition in a retaining α-l-arabinofuranosidase

“…Three main approaches have been identified to create TGs starting from rGHs [16]. The first involves the modification of substrate interactions in the donor subsite(s), leading to increased transition state (TS) energy barriers [26,27]. The aim is to reduce the efficiency of watermediated deglycosylation, thus favouring transglycosylation, although overall catalytic activity is also affected.…”

“…transglycosylation). This is often achieved by introducing hydrophobic amino acid side chains at the acceptor site(s), which form favourable interactions with incoming glycoside acceptors and repel water molecules [26,28,29]. When combined with donor subsite engineering, the introduction of favourable acceptor site interactions can partially compensate for the overall reduction in efficiency due to the poor formation of the glycosyl-enzyme intermediate [30].…”