Enzymes exist as a dynamic ensemble of conformations, each potentially playing a key role in substrate binding, the chemical transformation, or product release. We discuss recent advances in the evaluation of the enzyme conformational dynamics and its evolution towards new functions or substrate preferences.
Multimeric enzyme
complexes are ubiquitous in nature and catalyze
a broad range of useful biological transformations. They are often
characterized by a tight allosteric coupling between subunits, making
them highly inefficient when isolated. A good example is Tryptophan
synthase (TrpS), an allosteric heterodimeric enzyme in the form of
an αββα complex that catalyzes the biosynthesis
of L-tryptophan. In this study, we decipher the allosteric
regulation existing in TrpS from Pyrococcus furiosus (PfTrpS), and how the allosteric conformational
ensemble is recovered in laboratory-evolved stand-alone β-subunit
variants. We find that recovering the conformational ensemble of a
subdomain of TrpS affecting the relative stabilities of open, partially
closed, and closed conformations is a prerequisite for enhancing the
catalytic efficiency of the β-subunit in the absence of its
binding partner. The distal mutations resuscitate the allosterically
driven conformational regulation and alter the populations and rates
of exchange between these multiple conformational states, which are
essential for the multistep reaction pathway of the enzyme. Interestingly,
these distal mutations can be a priori predicted by careful analysis
of the conformational ensemble of the TrpS enzyme through computational
methods. Our study provides the enzyme design field with a rational
approach for evolving allosteric enzymes toward improved stand-alone
function for biosynthetic applications.
Allostery is a central
mechanism for the regulation of multi-enzyme
complexes. The mechanistic basis that drives allosteric regulation
is poorly understood but harbors key information for enzyme engineering.
In the present study, we focus on the tryptophan synthase complex
that is composed of TrpA and TrpB subunits, which allosterically activate
each other. Specifically, we develop a rational approach for identifying
key amino acid residues of TrpB distal from the active site. Those
residues are predicted to be crucial for shifting the inefficient
conformational ensemble of the isolated TrpB to a productive ensemble
through intra-subunit allosteric effects. The experimental validation
of the conformationally driven TrpB design demonstrates its superior
stand-alone activity in the absence of TrpA, comparable to those enhancements
obtained after multiple rounds of experimental laboratory evolution.
Our work evidences that the current challenge of distal active site
prediction for enhanced function in computational enzyme design has
become within reach.
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