The interaction of human galectin‐1 with a variety of oligosaccharides, from di‐(N‐acetyllactosamine) to tetra‐saccharides (blood B type‐II antigen) has been scrutinized by using a combined approach of different NMR experiments, molecular dynamics (MD) simulations, and isothermal titration calorimetry. Ligand‐ and receptor‐based NMR experiments assisted by computational methods allowed proposing three‐dimensional structures for the different complexes, which explained the lack of enthalpy gain when increasing the chemical complexity of the glycan. Interestingly, and independently of the glycan ligand, the entropy term does not oppose the binding event, a rather unusual feature for protein‐sugar interactions. CLEANEX‐PM and relaxation dispersion experiments revealed that sugar binding affected residues far from the binding site and described significant changes in the dynamics of the protein. In particular, motions in the microsecond‐millisecond timescale in residues at the protein dimer interface were identified in the presence of high affinity ligands. The dynamic process was further explored by extensive MD simulations, which provided additional support for the existence of allostery in glycan recognition by human galectin‐1.
Pharmaceutical industry is progressively replacing the chemical synthesis of cholesterol‐lowering agents by enzymatic processes. The directed evolution of acyltransferase LovD was a breakthrough in the synthesis of simvastatin, although little is known about how the in vitro evolution path raised up an engineered variant (LovD9) with excellent biocatalytic properties (high catalytic efficiency and stability under reaction conditions). In this study, we unveil how different mutation clusters scattered across LovD9 primary sequence specifically contribute to enhance both enzyme kinetics and stability. To this aim, simvastatin synthetic and hydrolytic activities, kinetic parameters and thermostability of several engineered variants were assessed. Through a rational combination of those clusters of mutations, we generated the variant LovD−BuCh2 whose catalytic efficiency is around 90 % of that obtained with LovD9 but with 15 less mutations. Supported by molecular dynamics simulations, this work demonstrates the cumulative effect of mutations at both the active site and the substrate entrance channel to enhance binding of the acyl donor and speed up the acyl transfer step from the acyl‐enzyme complex to monacolin J acid, while simultaneously minimizing detrimental side‐reaction pathways, substrate inhibition and increasing thermostability.
The
chemistry of diazo compounds has generated a huge breadth of
applications in the field of organic synthesis. Their versatility
combined with their tunable reactivity, stability, and chemoselectivity
makes diazo compounds desirable reagents for chemical biologists.
Here, we describe a method for the precise installation of diazo handles
on proteins and antibodies in a mild and specific approach. Subsequent
1,3-cycloaddition reactions with strained alkynes enable both bioimaging
through an in-cell “click” reaction and probing of the
cysteine proteome in cell lysates. The selectivity and efficiency
of these processes makes these suitable reagents for chemical biology
studies.
Laccases are in increasing demand
as innovative solutions in the
biorefinery fields. Here, we combine mutagenesis with structural,
kinetic, and in silico analyses to characterize the
molecular features that cause the evolution of a hyperthermostable
metallo-oxidase from the multicopper oxidase family into a laccase
(k
cat 273 s–1 for a
bulky aromatic substrate). We show that six mutations scattered across
the enzyme collectively modulate dynamics to improve the binding and
catalysis of a bulky aromatic substrate. The replacement of residues
during the early stages of evolution is a stepping stone for altering
the shape and size of substrate-binding sites. Binding sites are then
fine-tuned through high-order epistasis interactions by inserting
distal mutations during later stages of evolution. Allosterically
coupled, long-range dynamic networks favor catalytically competent
conformational states that are more suitable for recognizing and stabilizing
the aromatic substrate. This work provides mechanistic insight into
enzymatic and evolutionary molecular mechanisms and spots the importance
of iterative experimental and computational analyses to understand
local-to-global changes.
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