44 Foundational Undergraduate Experiences in the Laboratory Investigating the Role of F194T in the Bacterial Deacetylase LpxC

Authors:

Wessal Bakry, Cait Moffatt, Sien Verschave, Daniel Kahne

Date Created:

2025-01-01

Course Title:

Professor:

Not specified

About Paper:

The outer membrane of Gram-negative bacteria is a major mutated E. coli Phe194 to threonine (F194T) to assess whether contributor to their intrinsic antibiotic resistance due to its lowintroducing a smaller polar side chain in place of the native permeability and the presence of lipopolysaccharide (LPS), a hydrophobic group alters substrate binding or enzymatic activity. molecule essential for bacterial viability and pathogenesis. The We hypothesize that this substitution will disrupt active site cytoplasmic deacetylase LpxC catalyzes the first committed step geometry and reduce LpxC catalytic activity. Using PCR site- in LPS biosynthesis, making it a promising target for novel directed mutagenesis, we introduced the F194T mutation into a antibiotic development. Recent work has revealed the first crystal plasmid containing LpxC, which was subsequently transformed structure of LpxC bound to its natural reaction product, offering into E. coli. We are currently in the process of conducting studies a true snapshot of the enzyme in its functional state and revealingto further assess enzymatic activity and evaluate how this mutation key substrate interactions that guide product binding and inform affects LPS biosynthesis and bacterial growth. This work aims to antibiotic design. Building on this structure, we investigated deepen our mechanistic understanding of LpxC and contribute to the functional role of phenylalanine 194 (Phe194) in insert II of structure-guidedantibioticdesign. Asdrug-resistantinfectionsrise Escherichia coli LpxC—a residue shown in prior structural studies worldwide,thereisanurgentneedtoidentifynewantibiotictargets to interact with the glucosamine component of the substrate. We in essential bacterial pathways to improve therapeutic outcomes. Aminative Suzuki-Miyaura Cross-Coupling Colin Bloom, Paul Onnuch, Dilek Dogutan Kiper Harvard College | Mather House | Chemistry | 2028 The Suzuki-Miyaura cross-coupling (SMC) reaction is a widely hydroxylamine) were utilized in the same reaction. Inspired used method for forming carbon-carbon bonds through a boronic by their work, we are working on improving the synthesis by acid connected to an R group, an organohalide containing a incorporating microwave irradiation. Our preliminary results second R group, a base (usually K SO )2 an4 a Pd catalyst. indicate that Microwave-Assisted Aminative Suzuki-Miyaura Liu et al., have recently shown that these SMC reagents could reactions offer several advantages over conventional heating: (1) be utilized to facilitate “nitrene” insertion between two carbons depending on the substituents, there was a significant reduction allowing formation of C–N–C bonds. This strategy introduces a in reaction times from 12-48 hours to 6-12 hours under the novel synthetic methodology where industrial synthesis of many same catalyst conditions (3 mol % tBuBrettPhos–Pd), resulting products bearing a C–N–C motif becomes much less complicated, in energy and cost savings; (2) cleaner crude products due to and also utilizes the starting materials from a SMC reaction, fewer side reactions; and (3) easier purification, requiring less consequently reducing the total cost of the desired products. Liu solvent, silica, and other materials and supplies, which in turn et al. have successfully demonstrated that various substituents reduces laboratory waste and further cuts costs creating cleaner couldbeusedfortheAminativeSuzuki-Miyaurareactionincluding and safer chemistry research in general. These findings show that aryl halides and pseudohalides, boronic acids and esters if a bulkyusingmicrowaveirradiationwillbemoreeffectiveandefficientfor ancillary phosphine ligand on palladium (tBuBrettPhos) and a industrial applications, such as drug synthesis. commercially available amination reagent (O-diphenylphosphinyl A Visualization of Escherichia coli Cells with a H265A Mutation in the LpxC Enzyme Evan Carpenter, Cait Moffatt, Sien Verschave, Daniel Kahne Harvard College | Eliot House | Neuroscience | 2028 The LpxC enzyme is essential in the synthesis of with 5’-GCG-3’ to introduce the H265A mutation in E. coli. After lipopolysaccharide, a key component of the outer membrane our mutant plasmids were amplified, we transformed E. coli cells in gram negative bacteria, like Escherichia coli, making it an touptakethem. Wethenranproteinexpressiontests, usingvarying excellent target for novel antibiotics. This treatment potential hasmounts of IPTG to produce different amounts of wild-type LpxC inspired many people to make mutants of the protein, targeting key to determine optimal protein expression conditions for when we amino acid sites. Although many mutations have been introduced, purify our mutants. We ran our purified proteins on an SDS-PAGE many of them have not been visualized within the context of the gel. We plan to do the same approach with the mutant LpxC cell. We mutated H265, an amino acid important in the catalytic plasmids and overexpress them to purify the mutant protein. We activity of the enzyme, into an alanine, which may later lead to will then visualize both the mutant and wildtype cells to see how an inability for the cell to form the outer membrane. We designed the outer membrane compares between the two. If the bacterial PCR primers to amplify a plasmid containing the LpxC gene and cells cannot survive with the mutation, it could provide valuable the Lac operon. In the primers, we replaced the 5’-CAT-3’ codon information for the development of novel antibiotics.

Abstract:

The outer membrane of Gram-negative bacteria is a major mutated E. coli Phe194 to threonine (F194T) to assess whether contributor to their intrinsic antibiotic resistance due to its lowintroducing a smaller polar side chain in place of the native permeability and the presence of lipopolysaccharide (LPS), a hydrophobic group alters substrate binding or enzymatic activity. molecule essential for bacterial viability and pathogenesis. The We hypothesize that this substitution will disrupt active site cytoplasmic deacetylase LpxC catalyzes the first committed step geometry and reduce LpxC catalytic activity. Using PCR site- in LPS biosynthesis, making it a promising target for novel directed mutagenesis, we introduced the F194T mutation into a antibiotic development. Recent work has revealed the first crystal plasmid containing LpxC, which was subsequently transformed structure of LpxC bound to its natural reaction product, offering into E. coli. We are currently in the process of conducting studies a true snapshot of the enzyme in its functional state and revealingto further assess enzymatic activity and evaluate how this mutation key substrate interactions that guide product binding and inform affects LPS biosynthesis and bacterial growth. This work aims to antibiotic design. Building on this structure, we investigated deepen our mechanistic understanding of LpxC and contribute to the functional role of phenylalanine 194 (Phe194) in insert II of structure-guidedantibioticdesign. Asdrug-resistantinfectionsrise Escherichia coli LpxC—a residue shown in prior structural studies worldwide,thereisanurgentneedtoidentifynewantibiotictargets to interact with the glucosamine component of the substrate. We in essential bacterial pathways to improve therapeutic outcomes. Aminative Suzuki-Miyaura Cross-Coupling Colin Bloom, Paul Onnuch, Dilek Dogutan Kiper Harvard College | Mather House | Chemistry | 2028 The Suzuki-Miyaura cross-coupling (SMC) reaction is a widely hydroxylamine) were utilized in the same reaction. Inspired used method for forming carbon-carbon bonds through a boronic by their work, we are working on improving the synthesis by acid connected to an R group, an organohalide containing a incorporating microwave irradiation. Our preliminary results second R group, a base (usually K SO )2 an4 a Pd catalyst. indicate that Microwave-Assisted Aminative Suzuki-Miyaura Liu et al., have recently shown that these SMC reagents could reactions offer several advantages over conventional heating: (1) be utilized to facilitate “nitrene” insertion between two carbons depending on the substituents, there was a significant reduction allowing formation of C–N–C bonds. This strategy introduces a in reaction times from 12-48 hours to 6-12 hours under the novel synthetic methodology where industrial synthesis of many same catalyst conditions (3 mol % tBuBrettPhos–Pd), resulting products bearing a C–N–C motif becomes much less complicated, in energy and cost savings; (2) cleaner crude products due to and also utilizes the starting materials from a SMC reaction, fewer side reactions; and (3) easier purification, requiring less consequently reducing the total cost of the desired products. Liu solvent, silica, and other materials and supplies, which in turn et al. have successfully demonstrated that various substituents reduces laboratory waste and further cuts costs creating cleaner couldbeusedfortheAminativeSuzuki-Miyaurareactionincluding and safer chemistry research in general. These findings show that aryl halides and pseudohalides, boronic acids and esters if a bulkyusingmicrowaveirradiationwillbemoreeffectiveandefficientfor ancillary phosphine ligand on palladium (tBuBrettPhos) and a industrial applications, such as drug synthesis. commercially available amination reagent (O-diphenylphosphinyl A Visualization of Escherichia coli Cells with a H265A Mutation in the LpxC Enzyme Evan Carpenter, Cait Moffatt, Sien Verschave, Daniel Kahne Harvard College | Eliot House | Neuroscience | 2028 The LpxC enzyme is essential in the synthesis of with 5’-GCG-3’ to introduce the H265A mutation in E. coli. After lipopolysaccharide, a key component of the outer membrane our mutant plasmids were amplified, we transformed E. coli cells in gram negative bacteria, like Escherichia coli, making it an touptakethem. Wethenranproteinexpressiontests, usingvarying excellent target for novel antibiotics. This treatment potential hasmounts of IPTG to produce different amounts of wild-type LpxC inspired many people to make mutants of the protein, targeting key to determine optimal protein expression conditions for when we amino acid sites. Although many mutations have been introduced, purify our mutants. We ran our purified proteins on an SDS-PAGE many of them have not been visualized within the context of the gel. We plan to do the same approach with the mutant LpxC cell. We mutated H265, an amino acid important in the catalytic plasmids and overexpress them to purify the mutant protein. We activity of the enzyme, into an alanine, which may later lead to will then visualize both the mutant and wildtype cells to see how an inability for the cell to form the outer membrane. We designed the outer membrane compares between the two. If the bacterial PCR primers to amplify a plasmid containing the LpxC gene and cells cannot survive with the mutation, it could provide valuable the Lac operon. In the primers, we replaced the 5’-CAT-3’ codon information for the development of novel antibiotics.

Source:

Harvard / Howard University | Economics | 2027 / 2025

Topics:

lpxc, coli, reaction, mutation, cell, mutant, bacterial, antibiotic, product, outer, membrane, activity

Co-authors:

@wessalbakry188 , @caitmoffatt189 , @sienverschave190 , @danielkahne191