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Epistasis is a key factor in evolution since it determines which combinations of mutations provide adaptive solutions and which mutational pathways toward these solutions are accessible by natural selection. There is growing evidence for the pervasiveness of sign epistasis--a complete reversion of mutational effects, particularly in protein evolution--yet its molecular basis remains poorly understood. We describe the structural basis of sign epistasis between G238S and R164S, two adaptive mutations in TEM-1 β-lactamase--an enzyme that endows antibiotics resistance. Separated by 10 Å, these mutations initiate two separate trajectories toward increased hydrolysis rates and resistance toward second and third-generation cephalosporins antibiotics. Both mutations allow the enzyme's active site to adopt alternative conformations and accommodate the new antibiotics. By solving the corresponding set of crystal structures, we found that R164S causes local disorder whereas G238S induces discrete conformations. When combined, the mutations in 238 and 164 induce local disorder whereby nonproductive conformations that perturb the enzyme's catalytic preorganization dominate. Specifically, Asn170 that coordinates the deacylating water molecule is misaligned, in both the free form and the inhibitor-bound double mutant. This local disorder is not restored by stabilizing global suppressor mutations and thus leads to an evolutionary cul-de-sac. Conformational dynamism therefore underlines the reshaping potential of protein's structures and functions but also limits protein evolvability because of the fragility of the interactions networks that maintain protein structures.

Many Gram‐negative bacteria use N‐acyl‐L‐homoserine lactone (AHL) signals to coordinate phenotypes such as biofilm formation and virulence factor production. Quorum‐quenching enzymes, such as AHL acylases, chemically degrade these molecules which prevents signal reception by bacteria and inhibits undesirable biofilm‐related traits. These capabilities make acylases appealing candidates for controlling microbes, yet candidates with high activity levels and substrate specificity and that are capable of being formulated into materials are needed. In this work, we undertook engineering efforts against two AHL acylases, PvdQ and MacQ, to generate these improved properties using the Protein One‐Stop Shop Server. The engineering of acylases is complicated by low‐throughput enzymatic assays. Alleviating this challenge, we report a time‐course kinetic assay for AHL acylases that monitors the real‐time production of homoserine lactone. Using the assay, we identified variants of PvdQ that were significantly stabilized, with melting point increases of up to 13.2°C, which translated into high resistance against organic solvents and increased compatibility with material coatings. While the MacQ mutants were unexpectedly destabilized, they had considerably improved kinetic properties, with >10‐fold increases against N‐butyryl‐L‐homoserine lactone and N‐hexanoyl‐L‐homoserine lactone. Accordingly, these changes resulted in increased quenching abilities using a biosensor model and greater inhibition of virulence factor production of Pseudomonas aeruginosa PA14. While the crystal structure of one of the MacQ variants, M1, did not reveal obvious structural determinants explaining the observed changes in kinetics, it allowed for the capture of an acyl‐enzyme intermediate that confirms a previously hypothesized catalytic mechanism of AHL acylases. [ABSTRACT FROM AUTHOR]

Metformin is the first-line treatment for type II diabetes patients and a pervasive pollutant with more than 180 million kg ingested globally and entering wastewater. The drug's direct mode of action is currently unknown but is linked to effects on gut microbiomes and may involve specific gut microbial reactions to the drug. In wastewater treatment plants, metformin is known to be transformed by microbes to guanylurea, although genes encoding this metabolism had not been elucidated. In the present study, we revealed the function of two genes responsible for metformin decomposition (mfmA and mfmB) found in isolated bacteria from activated sludge. MfmA and MfmB form an active heterocomplex (MfmAB) and are members of the ureohydrolase protein superfamily with binuclear metal-dependent activity. MfmAB is nickel-dependent and catalyzes the hydrolysis of metformin to dimethylamine and guanylurea with a catalytic efficiency (kcat/KM) of 9.6 x 10³ M-1s-1 and KM for metformin of 0.82 mM. MfmAB shows preferential activity for metformin, being able to discriminate other close substrates by several orders of magnitude. Crystal structures of MfmAB show coordination of binuclear nickel bound in the active site of the MfmA subunit but not MfmB subunits, indicating that MfmA is the active site for the MfmAB complex. Mutagenesis of residues conserved in the MfmA active site revealed those critical to metformin hydrolase activity and its small substrate binding pocket allowed for modeling of bound metformin. This study characterizes the products of the mfmAB genes identified in wastewater treatment plants on three continents, suggesting that metformin hydrolase is widespread globally in wastewater. [ABSTRACT FROM AUTHOR]

Arsenate and phosphate are abundant on Earth and have striking similarities: nearly identical p[K.sub.a] values (1,2), similarly charged oxygen atoms, and thermochemical radii that differ by only 4% (ref. 3). [...]