# Structure and mechanism of membrane enzymes responsible for bacterial lipid modification and polymyxin resistance

> **NIH NIH R35** · RUTGERS BIOMEDICAL AND HEALTH SCIENCES · 2024 · $392,500

## Abstract

Project Summary/Abstract
 Antibiotic resistance is a rapidly growing threat to human health, further exacerbated by the limited
development of new antibiotics. Thus, there is a dire need for research informing the design of new therapeutic
options to counter the rise of antibiotic resistance. Polymyxins are cationic antimicrobial peptides that
associate with the outer membrane of Gram-negative (GN) bacteria through electrostatic interactions and are
considered the last line of defense against multi-drug resistant GN bacterial infections. Yet, resistance to
polymyxins develops often and with relative ease, due to modifications that bacteria have developed as
defenses against antimicrobial peptides (AMPs) produced by the innate immune system or secreted by other
bacterial species. Modification of Lipid A, the lipidic anchor of the bacterial lipopolysaccharide (LPS or
endotoxin) decorating the outer membrane of GN bacteria, with diverse chemical moieties, is a common
mechanism leading to resistance to antimicrobial agents. In E. coli, S. enterica and P. aeruginosa, “capping”
of the phosphates of Lipid A with an aminoarabinose moiety (L-Ara4N) is the predominant modification leading
to resistance against polymyxins and AMPs. The aminoarabinose “cap” is synthesized by GN bacteria through
an enzymatic relay of eight proteins collectively called the aminoarabinose biosynthetic pathway. The
mechanistic basis of function for the membrane enzymes of the pathway is poorly understood, in large part
due to the technical challenges associated with studying enzymes that function at or near the membrane and
utilize lipidic substrates. As part of this research program, we will use a variety of experimental techniques,
including cryo-electron microscopy (cryoEM), mutagenesis, bacterial growth assays, bacterial genetics, and
microscale thermophoresis (MST), to achieve the following core goals: (1) Structure determination and
substrate-binding characterization for the three bona fide membrane enzymes that operate in the
aminoarabinose biosynthetic pathway (the polyprenol phosphate glycosyltransferase ArnC, the deformylase
ArnD and the lipid-to-lipid glycosyltransferase ArnT), and (2) Investigating the mechanistic basis of enzymatic
function, metal cofactor coordination, and catalysis, in each of the three membrane enzymes under study.
The research program will leverage our multidisciplinary training in membrane protein biochemistry and
structural biology, and experience gained from having successfully solved several structures of the enzyme
ArnT bound to different lipidic substrates. The impact of the program lies within its potential to: i) Provide
detailed mechanistic insights into the structural basis of a diverse set of enzymatic functions responsible for
aminoarabinose biosynthesis and polymyxin resistance in GN bacteria, ii) Advance our understanding of
protein-lipid interactions with undecaprenyl phosphate, as all three enzymes under study utilize undecapreny...

## Key facts

- **NIH application ID:** 10881887
- **Project number:** 5R35GM150831-02
- **Recipient organization:** RUTGERS BIOMEDICAL AND HEALTH SCIENCES
- **Principal Investigator:** Vasileios I Petrou
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $392,500
- **Award type:** 5
- **Project period:** 2023-07-10 → 2028-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10881887

## Citation

> US National Institutes of Health, RePORTER application 10881887, Structure and mechanism of membrane enzymes responsible for bacterial lipid modification and polymyxin resistance (5R35GM150831-02). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10881887. Licensed CC0.

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