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Review
. 2025 Aug 19;64(16):3495-3507.
doi: 10.1021/acs.biochem.5c00165. Epub 2025 Aug 5.

Considering Metabolic Context in Enzyme Evolution and Design

Dhiraj Dokwal  1 , Philip M Brown  1 , Karolina Filipowska  1   2 , Kimberly A Reynolds  1   2
Affiliations
Review

Considering Metabolic Context in Enzyme Evolution and Design

Dhiraj Dokwal et al. Biochemistry. .

Abstract

Enzymes are often treated as isolated molecular entities when measuring their biochemical activity and designing synthetic sequences. Yet inside the cell, enzymes interact through shared metabolite pools, physical binding, and regulatory feedback. This coordination is necessary for responsive, cohesive metabolic behavior. Moreover, these interactions place constraints on enzyme activity, specificity, abundance, and ultimately sequence. Defining these interaction-mediated constraints is important to understand metabolic evolution and to design synthetic systems. For example, the influence of the cellular and environmental milieu becomes evident when a heterologous enzyme from one species fails to function properly upon transfer to a new host. In this review we consider how cell context shapes enzyme evolution, and in turn, how variations in enzyme biochemistry, biophysics, and abundance impact cell fitness.

Keywords: CRISPR; Escherichia coli; NADPH; deep mutational scanning; dihydrofolate reductase.

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