Dataset Information

Dissecting enzyme function with microfluidic-based deep mutational scanning.

ABSTRACT: Natural enzymes are incredibly proficient catalysts, but engineering them to have new or improved functions is challenging due to the complexity of how an enzyme's sequence relates to its biochemical properties. Here, we present an ultrahigh-throughput method for mapping enzyme sequence-function relationships that combines droplet microfluidic screening with next-generation DNA sequencing. We apply our method to map the activity of millions of glycosidase sequence variants. Microfluidic-based deep mutational scanning provides a comprehensive and unbiased view of the enzyme function landscape. The mapping displays expected patterns of mutational tolerance and a strong correspondence to sequence variation within the enzyme family, but also reveals previously unreported sites that are crucial for glycosidase function. We modified the screening protocol to include a high-temperature incubation step, and the resulting thermotolerance landscape allowed the discovery of mutations that enhance enzyme thermostability. Droplet microfluidics provides a general platform for enzyme screening that, when combined with DNA-sequencing technologies, enables high-throughput mapping of enzyme sequence space.

SUBMITTER: Romero PA

PROVIDER: S-EPMC4466731 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Dissecting enzyme function with microfluidic-based deep mutational scanning.

Romero Philip A PA Tran Tuan M TM Abate Adam R AR

Proceedings of the National Academy of Sciences of the United States of America 20150526 23

Natural enzymes are incredibly proficient catalysts, but engineering them to have new or improved functions is challenging due to the complexity of how an enzyme's sequence relates to its biochemical properties. Here, we present an ultrahigh-throughput method for mapping enzyme sequence-function relationships that combines droplet microfluidic screening with next-generation DNA sequencing. We apply our method to map the activity of millions of glycosidase sequence variants. Microfluidic-based de ...[more]

PMID: 26040002

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Project description:Colistin (polymyxin E) and polymyxin B have been used as last-resort agents for treating infections caused by multidrug-resistant Gram-negative bacteria. However, their efficacy has been challenged by the emergence of the mobile colistin resistance gene mcr-1, which encodes a transmembrane phosphoethanolamine (PEA) transferase enzyme, MCR-1. The enzyme catalyzes the transfer of the cationic PEA moiety of phosphatidylethanolamine (PE) to lipid A, thereby neutralizing the negative charge of lipid A and blocking the binding of positively charged polymyxins. This study aims to facilitate understanding of the mechanism of the MCR-1 enzyme by investigating its active-site sequence requirements. For this purpose, 23 active-site residues of MCR-1 protein were randomized by constructing single-codon randomization libraries. The libraries were individually selected for supporting Escherichia coli cell growth in the presence of colistin or polymyxin B. Deep sequencing of the polymyxin-resistant clones revealed that wild-type residues predominates at 17 active-site residue positions, indicating these residues play critical roles in MCR-1 function. These residues include Zn2+-chelating residues as well as residues that may form a hydrogen bond network with the PEA moiety or make hydrophobic interactions with the acyl chains of PE. Any mutations at these residues significantly decrease polymyxin resistance levels and the PEA transferase activity of the MCR-1 enzyme. Therefore, deep sequencing of the randomization libraries of MCR-1 enzyme identifies active-site residues that are essential for its polymyxin resistance function. Thus, these residues may be utilized as targets to develop inhibitors to circumvent MCR-1-mediated polymyxin resistance. IMPORTANCE Polymyxin antibiotics are used as last-line antibiotics in treating infections caused by multidrug-resistant pathogens. However, widespread use of polymyxins has led to the emergence of resistance. Although multiple mechanisms for resistance exist, that due to mcr-1 is a particular concern, as it can be readily transferred among bacterial pathogens. The mcr-1 gene encodes a transmembrane phosphoethanolamine (PEA) transferase that modifies lipid A to block the binding of polymyxin antibiotics. We utilized random mutagenesis coupled with next-generation sequencing to determine the amino acid sequence requirements of 23 residues in and near the active site of MCR-1. We show that the enzyme has stringent sequence requirements, with 75% of the residues examined being essential for function. Coupled with the finding that these residues are largely conserved among PEA enzymes, the results suggest inhibitors that bind near these sites will broadly inhibit MCR-1 and other enzymes of this class.

Dataset Information

Dissecting enzyme function with microfluidic-based deep mutational scanning.

Publications

Dissecting enzyme function with microfluidic-based deep mutational scanning.

Similar Datasets

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