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Title page for ETD etd-09152014-151801

Type of Document Dissertation
Author Xiao, Yi
URN etd-09152014-151801
Title Functional annotation of orphan human P450 enzymes: heterologous expression and substrate searches by metabolomic approaches
Degree PhD
Department Biochemistry
Advisory Committee
Advisor Name Title
F. Peter Guengerich Committee Chair
Alan Brash Committee Member
David Hachey Committee Member
Kevin Schey Committee Member
Richard Armstrong Committee Member
  • functional annotation
  • substrate searches
  • orphan human P450 enzyme
  • metabolomic approaches
Date of Defense 2014-09-10
Availability unrestricted
Cytochrome P450s are versatile biocatalysts that play crucial roles in many important biological processes. Functional annotation of orphan P450 enzymes is the focus of this work.

Catalytic activities of orphan human P450 2S1, 2W1, and 4X1 were investigated with untargeted metabolomic approaches. A series of lysophospholipids and free fatty acids were identified as novel substrates for P450 2W1. The isomer- and enantiomer-selectivity of P450 2W1-catalyzed lysophospholipid oxidations were characterized. The identities of the oxidation products were defined, and steady-state kinetics of the P450 reactions were determined. Epoxidation and hydroxylation of 18:1 lysophospholipid are considerably more efficient than that of the 18:1 free fatty acid. No endogenous substrate was identified for human P450 2S1 or 4X1.

Heterologous expression systems for human P450 2U1, 4V2, 4Z1, and 46A1 were established for functional studies.

A new software system was established to search for isotopic patterns in LC-MS data. It was used to identify monooxygenated enzymatic products after incubation with 16O2/18O2 gas.

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