Structural Characterization of the Novel Nitrating Enzyme RufO Restricted; Files Only

Abstract

Cytochrome P450 enzymes are a diverse class of heme-containing metalloenzymes that most commonly catalyze monooxygenation reactions in metabolism and natural product biosynthesis. Recently, a small subset of noncanonical P450 enzymes has been discovered that catalyze direct aromatic nitration reactions, an uncommon biological transformation. RufO, a cytochrome P450 from Streptomyces atratus, catalyzes the nitration of tyrosine to produce 3-nitrotyrosine, a non-proteinogenic amino acid incorporated into the anti-mycobacterial natural product rufomycin. Despite the importance of this transformation, the structural determinants governing substrate recognition and catalysis by RufO remain poorly understood. Here, structural approaches were used to investigate substrate binding and catalysis by RufO. A crystal structure of RufO in complex with the peptide substrate MRYLH was solved using X-ray crystallography, revealing a network of hydrogen bonding and hydrophobic interactions that position the tyrosine residue above the heme cofactor. Comparison with the previously determined substrate-free structure shows that substrate binding induces ordering of the FG loop into a closed-lid conformation that encloses the active site. Additional crystal structures with substrate analogs demonstrate that RufO can accommodate certain substitutions on the aromatic ring without significantly altering substrate orientation, although these analogs do not undergo turnover, indicating that proper positioning alone is insufficient for catalysis. To further probe residues involved in substrate recognition, the H170Y mutant of RufO was generated and characterized. To complement these structural studies, a custom Python-based bioinformatic pipeline was developed to search genomic data for potential novel nitrating P450 enzymes by identifying biosynthetic gene clusters containing a cytochrome P450, a nitric oxide synthase, and a short peptide motif. Although no new nitrating enzymes were identified, the pipeline successfully recovered known nitrating enzymes and establishes a framework for future mining efforts. Together, these studies provide new structural insights into substrate recognition by RufO and introduce computational tools for identifying additional nitrating enzymes.

Table of Contents

Introduction 1

   Cytochrome P450s and Nitrating Enzymes 1

   Elucidation of the True Substrate of RufO 4

   Potential Outcomes and Implications 5

Aim I 6

Methods I 8

   Sequencing Analysis 8

   Test Expression 8

   Co-Expression with Chaperone Systems 11

   RufO H170Y Expression 14

   RufO H170Y Lysis and Purification 14

   Protein Crystallization 17

   Protein X-ray Crystallography 19

Results I 20

Aim II 22

Methods II 24

   Crystallizing RufO with Substrate Analogs 24

   Capturing Reaction Intermediates 25

Results II 26

Aim III 35

Methods III 36

Results III 38

Conclusion 41

References 43

Appendix 48

   pipeline1.py 48

   pipeline2.py 50

   pipeline3.py 52

About this Honors Thesis

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School	Emory College
Department	Chemistry
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Chemistry, Biochemistry
Keyword	natural product biosynthesis cytochrome P450s X-ray crystallography nitration RufO
Committee Chair / Thesis Advisor	Katherine Davis, Emory University
Committee Members	Richard Himes, Emory University Christine Dunham, Emory University

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