Reduced Processivity in a Chitobiohydrolase Enhances LPMO-Assisted Chitin Depolymerization

TL;DR

Reducing the processivity of a chitin-degrading enzyme improves its performance when working with a helper enzyme, offering insights into efficient breakdown of tough sugars.

Contribution

Engineering a less processive chitobiohydrolase variant that outperforms the wild-type when paired with LPMOs.

Findings

Less processive SmChiB mutants, especially W220Y, showed improved performance with LPMOs.

Molecular dynamics confirmed Trp220's role in substrate binding and processivity.

Combining W220Y with LPMOs doubled soluble product yield compared to wild-type under identical conditions.

Abstract

Efficient enzymatic depolymerization of recalcitrant
polysaccharides
such as chitin and cellulose relies on processive glycoside hydrolases
(GHs), whose efficiency can be enhanced through cooperation with lytic
polysaccharide monooxygenases (LPMOs). For processive GHs, retained
binding during successive catalytic events aids depolymerization of
crystalline substrates but comes at the expense of slow dissociation
(low k
off) that limits turnover. Here,
we engineered a series of mutants of the processive exochitobiohydrolase SmChiB from Serratia marcescens, in which a major determinant of substrate affinity and processivity,
Trp220, was replaced by Tyr, Phe, His, Gln, or Ala. Functional analysis
showed that these mutants had stepwise reductions in substrate affinity
and processivity, with the latter being a signature of an increased k
off. Molecular dynamics simulations confirmed
that Trp220 plays an important role in substrate binding. The less
processive SmChiB variants, and in particular W220Y,
were able to reach wild-type-like performance only at high substrate
concentrations. Importantly, the use of LPMOs to decrystallize the
substrate and thereby increase its effective concentration, enhanced
the performance of the less processive mutants to a much greater extent
than for wild-type SmChiB. In some reaction setups,
the combination of W220Y with an LPMO yielded twice as much soluble
product compared to the wild-type enzyme under identical conditions.
Thus, when combined with LPMOs, less processive GHs become more favorable
because they are intrinsically more efficient catalysts when acting
on noncrystalline substrates. These findings shed light on how the
interplay between GHs and LPMOs can be optimized for efficient enzymatic
conversion of recalcitrant polysaccharides.

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