Understanding the molecular determinants of enzyme performance is of primary importance for the rational design of ad hoc mutants. A novel approach, which combines efficient conformational sampling and quick reactivity scoring, is used here to shed light on how substrate oxidation was improved during the directed evolution experiment of a fungal laccase (from Pycnoporus cinnabarinus), an industrially relevant class of oxidoreductases. It is found that the enhanced activity of the evolved enzyme is mainly the result of substrate arrangement in the active site, with no important change in the redox potential of the T1 copper. Mutations at the active site shift the binding mode into a more buried substrate position and provide a more favorable electrostatic environment for substrate oxidation. As a consequence, engineering the binding event seems to be a viable way to in silico evolution of oxidoreductases.
Through the application of a region-focused saturation mutagenesis and randomization approach, protein engineering of the Cal-A enzyme was undertaken with the goal of conferring new triglyceride selectivity. Little is known about the mode of triglyceride binding to Cal-A. Engineering Cal-A thus requires a systemic approach. Targeted and randomized Cal-A libraries were created, recombined using the Golden Gate approach and screened to detect variants able to discriminate between long-chain (olive oil) and short-chain (tributyrin) triglyceride substrates using a high-throughput in vivo method to visualize hydrolytic activity. Discriminative variants were analyzed using an in-house script to identify predominant substitutions. This approach allowed identification of variants that exhibit strong discrimination for the hydrolysis of short-chain triglycerides and others that discriminate towards hydrolysis of long-chain triglycerides. A clear pattern emerged from the discriminative variants, identifying the 217–245 helix-loop-helix motif as being a hot-spot for triglyceride recognition. This was the consequence of introducing the entire mutational load in selected regions, without putting a strain on distal parts of the protein. Our results improve our understanding of the Cal-A lipase mode of action and selectivity. This holistic perspective to protein engineering, where parts of the gene are individually mutated and the impact evaluated in the context of the whole protein, can be applied to any protein scaffold.
Whole-genome sequencing of trimethoprim-resistant Escherichia coli clinical isolates identified a member of the trimethoprim-resistant type II dihydrofolate reductase gene family (dfrB). The dfrB4 gene was located within a class I integron flanked by multiple resistance genes. This arrangement was previously reported in a 130.6-kb multiresistance plasmid. The DfrB4 protein conferred a Ͼ2,000-fold increased trimethoprim resistance on overexpression in E. coli. Our results are consistent with the finding that dfrB4 contributes to clinical trimethoprim resistance.KEYWORDS type II dihydrofolate reductase, trimethoprim resistance, E. coli clinical isolates, dfrB4, antibiotic-resistant genes, class I integron, urinary tract infection P ublic health agencies worldwide rank trimethoprim (TMP) a broad-spectrum antibiotic of importance in human medicine (1). Widely used as a result of its low cost and effectiveness, TMP inhibits the activity of many microbial chromosomal dihydrofolate reductases (DHFRs); thus, DHFRs have long served as prioritized targets of antiproliferative drugs (2). Although the majority of living cells harbor a chromosomal member of the type I DHFR family, encoded by a dfrA homolog, the dfrB genes encode a family of plasmid-borne type II DHFRs that are evolutionarily unrelated to type I DHFRs. The dfrB genes have been found in pathogenic bacteria recovered from many food sources, including fish (3), pigs (4, 5), and cows (6), where they confer TMP resistance. Bacteria carrying dfrB genes have also been identified in wastewater samples (7). Over the past decade, dfrB genes have been tracked indirectly in antibiotic resistance studies through identification of integron-related elements (8-10). Therefore, the importance of dfrB genes in TMP resistance in human pathogens may be underappreciated (11,12).To date, only seven members of the dfrB gene family are known, and they are highly homologous (77% to 94% genetic identity, 77% to 99% amino acid identity) ( Table 1). Among these, the DfrB1 protein (also known as R67 DHFR) is the best-studied type II DHFR (13)(14)(15)(16)(17). It is proposed to be recently evolved, and it confers a significant survival advantage under TMP exposure to microbes that harbor it (18). To date, the family of dfrB genes has consistently been reported to be contained within the following mobile
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