Biology subjects

Gilman, M. S. A.

Publications and source records attributed to Gilman, M. S. A..

2 recordsLinked to original sources

bioRxiv2021

Biochemical and Structural Characterization of Two Cif-Like Epoxide Hydrolases from Burkholderia cenocepacia

Epoxide hydrolases catalyze the conversion of epoxides to vicinal diols in a range of cellular processes such as signaling, detoxification, and virulence. These enzymes typically utilize a pair of tyrosine residues to orient the substrate epoxide ring in the active site and stabilize the hydrolysis intermediate. A new subclass of epoxide hydrolases that utilize a histidine in place of one of the tyrosines was established with the discovery of the CFTR Inhibitory Factor (Cif) from Pseudomonas aeruginosa. Although the presence of such Cif-like epoxide hydrolases was predicted in other opportunistic pathogens based on sequence analyses, only Cif and its homologue aCif from Acinetobacter nosocomialis have been characterized. Here we report the biochemical and structural characteristics of Cfl1 and Cfl2, two Cif-like epoxide hydrolases from Burkholderia cenocepacia. Cfl1 is able to hydrolyze xenobiotic as well as biological epoxides that might be encountered in the environment or during infection. In contrast, Cfl2 shows very low activity against a diverse set of epoxides. The crystal structures of the two proteins reveal quaternary structures that build on the well-known dimeric assembly of the /{beta} hydrolase domain, but broaden our understanding of the structural diversity encoded in novel oligomer interfaces. Analysis of the interfaces reveals both similarities and key differences in sequence conservation between the two assemblies, and between the canonical dimer and the novel oligomer interfaces of each assembly. Finally, we discuss the effects of these higher-order assemblies on the intra-monomer flexibility of Cfl1 and Cfl2 and their possible roles in regulating enzymatic activity.

biochemistry↗

bioRxiv2020

Rapid generation of potent antibodies by autonomous hypermutation in yeast

The predominant approach for antibody generation remains animal immunization, which can yield exceptionally selective and potent antibody clones owing to the powerful evolutionary process of somatic hypermutation. However, animal immunization is inherently slow, has poor compatibility with certain antigens (e.g., integral membrane proteins), and suffers from self-tolerance and immunodominance, which limit the functional spectrum of antibodies that can be obtained. Here, we describe Autonomous Hypermutation yEast surfAce Display (AHEAD), a synthetic recombinant antibody generation technology that imitates somatic hypermutation inside engineered yeast. In AHEAD, antibody fragments are encoded on an error-prone orthogonal DNA replication system, resulting in Saccharomyces cerevisiae populations that continuously mutate surface-displayed antibody repertoires. Simple cycles of yeast culturing and enrichment for antigen binding drive the evolution of high-affinity antibody clones in a readily parallelizable process that takes as little as 2 weeks. We applied AHEAD to generate nanobodies against the SARS-CoV-2 S glycoprotein, a GPCR, and other targets. The SARS-CoV-2 nanobodies, concurrently evolved from an open-source naive nanobody library in 8 independent experiments, reached subnanomolar affinities through the sequential fixation of multiple mutations over 3-8 AHEAD cycles that saw [~]580-fold and [~]925-fold improvements in binding affinities and pseudovirus neutralization potencies, respectively. These experiments highlight the defining speed, parallelizability, and effectiveness of AHEAD and provide a template for streamlined antibody generation at large with salient utility in rapid response to current and future viral outbreaks.

bioengineering↗