# Mechanistic studies to enable rational design Class D monooxygenases

> **NIH NIH SC3** · CALIFORNIA STATE UNIVERSITY NORTHRIDGE · 2021 · $108,750

## Abstract

PROJECT SUMMARY / ABSTRACT
 With the emergence of bacterial resistance, identification of new diseases, and the need for new
therapeutics with different efficacies, our ability to design new drugs is becoming a more urgent priority.
Natural products are often useful as therapeutics for humans, though problems such as side effects and
production difficulties can preclude their successful development. This proposal seeks to enable development
of therapeutics through synthetic biology methods, in which the molecule's biosynthetic pathway is engineered
in order to alter the product. The Class D flavin monooxygenases are found in numerous natural product
biosynthetic pathways, including those of valanimycin and daunorubicin, two medicinally useful natural
products. With this research we hope to make the Class D flavin monooxygenases of these representative
biosynthetic pathways amenable to engineering for synthetic biology purposes.
 The enzyme-catalyzed step of interest here is a biosynthetic step common to multiple natural products
– flavin-dependent hydroxylation of a primary amine. The enzymes responsible for this step in the two
biosynthetic pathways – vlmH and DnmZ, respectively – will be biochemically characterized using transient-
state kinetics. Site-directed mutagenesis of active site residues will be combined with enzymatic activity and
binding studies to validate mechanistic steps and substrate binding interactions. The effect of changes in the
substrate binding site on the kinetics of intermediate formation will be investigated for use in validating
modifications to the enzyme's substrate specificity. The data yielded will enable rational design of vlmH and
DnmZ to alter their substrate binding preferences. Similar studies can be applied to other enzymes of the
pathways to introduce diversity into the molecules' final structures.

## Key facts

- **NIH application ID:** 10224872
- **Project number:** 5SC3GM122652-04
- **Recipient organization:** CALIFORNIA STATE UNIVERSITY NORTHRIDGE
- **Principal Investigator:** Jessica Vey
- **Activity code:** SC3 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $108,750
- **Award type:** 5
- **Project period:** 2018-09-15 → 2024-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10224872

## Citation

> US National Institutes of Health, RePORTER application 10224872, Mechanistic studies to enable rational design Class D monooxygenases (5SC3GM122652-04). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10224872. Licensed CC0.

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