Mapping enzyme catalysis with metabolomic biosensing

Enzymes are represented across a vast space of protein sequences and structural forms and have activities that far exceed the best chemical catalysts; however, engineering them to have novel or enhanced activity is limited by technologies for sensing product formation. Here, we describe a general and scalable approach for characterizing enzyme activity that uses the metabolism of the host cell as a biosensor by which to infer product formation. Since different products consume different molecules in their synthesis, they perturb host metabolism in unique ways that can be measured by mass spectrometry. This provides a general way by which to sense product formation, to discover unexpected products and map the effects of mutagenesis.

Download Full-text

Mapping enzyme catalysis with metabolic biosensing

Nature Communications ◽

10.1038/s41467-021-27185-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Linfeng Xu ◽

Kai-Chun Chang ◽

Emory M. Payne ◽

Cyrus Modavi ◽

Leqian Liu ◽

...

Keyword(s):

Mass Spectrometry ◽

Enzyme Activity ◽

Host Cell ◽

Enzyme Catalysis ◽

Protein Sequences ◽

Product Formation ◽

Enhanced Activity ◽

Host Metabolism

AbstractEnzymes are represented across a vast space of protein sequences and structural forms and have activities that far exceed the best chemical catalysts; however, engineering them to have novel or enhanced activity is limited by technologies for sensing product formation. Here, we describe a general and scalable approach for characterizing enzyme activity that uses the metabolism of the host cell as a biosensor by which to infer product formation. Since different products consume different molecules in their synthesis, they perturb host metabolism in unique ways that can be measured by mass spectrometry. This provides a general way by which to sense product formation, to discover unexpected products and map the effects of mutagenesis.

Download Full-text

Review for "A Novel Approach to Evaluate ELISA Antibody Coverage of Host Cell Proteins ‐ Combining ELISA‐based Immunocapture and Mass Spectrometry"

10.1002/btpr.2983/v1/review2 ◽

2019 ◽

Keyword(s):

Mass Spectrometry ◽

Host Cell ◽

Host Cell Proteins ◽

Novel Approach

Download Full-text

Brucella abortusinduces a Warburg shift in host metabolism that is linked to enhanced intracellular survival of the pathogen

10.1101/120527 ◽

2017 ◽

Author(s):

Daniel M. Czyż ◽

Jonathan Willett ◽

Sean Crosson

Keyword(s):

Lactic Acid ◽

Host Cell ◽

Brucella Abortus ◽

Cell Metabolism ◽

Lactate Production ◽

Human Macrophage ◽

Metabolic Shift ◽

Intracellular Infections ◽

Host Metabolism

ABSTRACTIntracellular bacterial pathogens exploit host cell resources to replicate and survive inside the host. Targeting these host systems is one promising approach to developing novel antimicrobials to treat intracellular infections. We show that human macrophage-like cells infected withBrucella abortusundergo a metabolic shift characterized by attenuated tricarboxylic acid cycle metabolism, reduced amino acid consumption, altered mitochondrial localization, and increased lactate production. This shift to an aerobic glycolytic state resembles the Warburg effect, a change in energy production that is well-described in cancer cells, and also occurs in activated inflammatory cells.B. abortusefficiently uses lactic acid as its sole carbon and energy source and requires the ability to metabolize lactate for normal survival in human macrophage-like cells. We demonstrate that chemical inhibitors of host glycolysis and lactate production do not affectin vitrogrowth ofB. abortusin axenic culture, but decrease its survival in the intracellular niche. Our data support a model in which infection shifts host metabolism to a Warburg-like state, andB. abortususes this change in metabolism to promote intracellular survival. Pharmacological perturbation of these features of host cell metabolism may be a useful strategy to inhibit infection by intracellular pathogens.IMPORTANCEBrucellaspp. are intracellular bacterial pathogens that cause disease in a range of mammals, including livestock. Transmission from livestock to humans is common and can lead to chronic human disease. Human macrophage-like cells infected withBrucella abortusundergo a Warburg-like metabolic shift to an aerobic glycolytic state where the host cells produce lactic acid and have reduced amino acid catabolism. We provide evidence that the pathogen can exploit this change in host metabolism to support growth and survival in the intracellular niche. Drugs that inhibit this shift in host cell metabolism inhibit intracellular replication and decrease the survival ofB. abortusin anin vitroinfection model; these drugs may be broadly useful therapeutics for intracellular infections.

Download Full-text