Epistasis between promoter activity and coding mutations shapes gene evolvability.

The evolution of protein-coding genes proceeds as mutations act on two main dimensions: regulation of transcription level and the coding sequence. The extent and impact of the connection between these two dimensions are largely unknown because they have generally been studied independently. By measuring the fitness effects of all possible mutations on a protein complex at various levels of promoter activity, we show that promoter activity at the optimal level for the wild-type protein masks the effects of both deleterious and beneficial coding mutations. Mutations that are deleterious at low activity but masked at optimal activity are slightly destabilizing for individual subunits and binding interfaces. Coding mutations that increase protein abundance are beneficial at low expression but could potentially incur a cost at high promoter activity. We thereby demonstrate that promoter activity in interaction with protein properties can dictate which coding mutations are beneficial, neutral, or deleterious.

A conserved SH3-like fold in diverse putative proteins tetramerizes into an oxidoreductase providing an antimicrobial resistance phenotype.

We present a potential mechanism for emergence of catalytic activity that is essential for survival, from a non-catalytic protein fold. The type B dihydrofolate reductase (DfrB) family of enzymes were first identified in pathogenic bacteria because their dihydrofolate reductase activity is sufficient to provide trimethoprim (TMP) resistance. DfrB enzymes are described as poorly evolved as a result of their unusual structural and kinetic features. No characterized protein shares sequence homology with DfrB enzymes; how they evolved to emerge in the modern resistome is unknown. In this work, we identify DfrB homologues from a database of putative and uncharacterized proteins. These proteins include an SH3-like fold homologous to the DfrB enzymes, embedded in a variety of additional structural domains. By means of functional, structural and biophysical characterization, we demonstrate that these distant homologues and their extracted SH3-like fold can display dihydrofolate reductase activity and confer TMP resistance. We provide evidence of tetrameric assembly and catalytic mechanism analogous to that of DfrB enzymes. These results contribute, to our knowledge, the first insights into a potential evolutionary path taken by this SH3-like fold to emerge in the modern resistome following introduction of TMP. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

Discovery of Highly Trimethoprim-Resistant DfrB Dihydrofolate Reductases in Diverse Environmental Settings Suggests an Evolutionary Advantage Unrelated to Antibiotic Resistance.

Type B dihydrofolate reductases (DfrB) are intrinsically highly resistant to the widely used antibiotic trimethoprim, posing a threat to global public health. The ten known DfrB family members have been strongly associated with genetic material related to the application of antibiotics. Several dfrB genes were associated with multidrug resistance contexts and mobile genetic elements, integrated both in chromosomes and plasmids. However, little is known regarding their presence in other environments. Here, we investigated the presence of dfrB beyond the traditional areas of enquiry by conducting metagenomic database searches from environmental settings where antibiotics are not prevalent. Thirty putative DfrB homologues that share 62 to 95% identity with characterized DfrB were identified. Expression of ten representative homologues verified trimethoprim resistance in all and dihydrofolate reductase activity in most. Contrary to samples associated with the use of antibiotics, the newly identified dfrB were rarely associated with mobile genetic elements or antibiotic resistance genes. Instead, association with metabolic enzymes was observed, suggesting an evolutionary advantage unrelated to antibiotic resistance. Our results are consistent with the hypothesis that multiple dfrB exist in diverse environments from which dfrB were mobilized into the clinically relevant resistome. Our observations reinforce the need to closely monitor their progression.

Glutamine-walking: Creating reactive substrates for transglutaminase-mediated protein labeling.

Chemically modified proteins are increasingly being tested and approved as therapeutic products. Batch-to-batch homogeneity is crucial to ensure safety and quality of therapeutic products. Highly selective protein modification may be achieved using enzymatic routes. Microbial transglutaminase (mTG) is a robust, easy to use and well-established enzyme that is used at a very large scale in the food industry such that its efficacy and its safety for human consumption are well established. In the context of therapeutic protein modification, mTG should crosslink one or more glutamines on the target protein with an aminated moiety such as a solubilizer, a tracer or a cytotoxic moiety. mTG has the advantage of being unreactive toward the majority of surface-exposed glutamines on most proteins, reducing sample heterogeneity. The caveat is that there may be no reactive glutamine on the target protein, or else a reactive glutamine may be found in a location where its modification compromises function of the target protein. Here we describe the glutamine-walk (Gln-walk), a straightforward method to create a glutamine-substrate site that is reactive to mTG in a target protein. Iterative substitution of single amino acids to a glutamine is followed by facile identification of reactivity with mTG, where covalent labeling of the target with an aminated fluorophore allows visualization of the most reactive modified targets. The approach is empirical; knowledge of the target protein structure and functional regions facilitates application of the method.