Toward a mechanistic understanding of cofactor biogenesis and catalysis in copper amine oxidases from Hansenula polymorpha.
2012-06
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Toward a mechanistic understanding of cofactor biogenesis and catalysis in copper amine oxidases from Hansenula polymorpha.
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2012-06
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Abstract
Copper amine oxidases (CAOs) are a ubiquitous class of enzymes responsible for
the oxidation of primary amines to their corresponding aldehydes with the concomitant
reduction of O2. Found in nearly every aerobic organism, CAOs take on a myriad of
roles depending on source, cellular location, and physiological substrate. In addition to
their catalytic activity, CAOs also produce their own redox-active cofactor, called 2,4,5-
trihydroxyphenylalanine quinone (TPQ), from an endogenous tyrosine residue. This
process occurs without additional enzymatic activity and requires only oxygen and
copper.
Two CAO paralogs from the yeast Hansenula polymorpha have been used for
structural studies aimed at deciphering mechanistic details of metal binding in TPQ
biogenesis as well as amine substrate specificity during catalysis. The X-ray crystal
structures of metal-free HPAO-1 (apoHPAO-1) as well as apoHPAO-1 in complex with
Cu(I) and Co(II) have been solved and are described in Chapter 3 of this work. Analysis
of these complexes has shed light into how different coordination geometry may
influence whether a metal is capable of initiating biogenesis in apoCAO.
In order to investigate amine substrate specificity in CAOs, the structure of a
second CAO from H. polymorpha (HPAO-2) has been solved and is presented in Chapter
2 of this work along with steady-state kinetic data for the reaction of HPAO-2 with
methylamine and benzylamine. HPAO-2 preferentially oxidizes benzylamine over
methylamine, while HPAO-1 prefers methylamine. Structural differences in the amine substrate channels of these two paralogs begin to account for their inverted substrate
preferences. Amine substrate specificity is further investigated in crystal structures of
HPAO-1 in complex with methylamine, ethylamine, and benzylamine. Substrate-protein
interactions in these three complexes shed light into this CAO’s preference for aliphatic
amines over the aromatic amines preferred by some of its homologs.
Finally, Chapter 5 of this work outlines two projects currently underway. The
first involves single site mutations in HPAO-1 and HPAO-2 at a position in the primary
sequence thought to contribute to substrate selection. An additional ongoing project involves solving the crystal structures of two aniline-substituted HPAO-1 proteins to
examine the factors governing the formation of a proposed Cu(I)/tyrosyl radical complex
during biogenesis.
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University of Minnesota Ph.D. dissertation. June 2012. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: Carrie M. Wilmot. 1 computer file (PDF); xii, 184 pages, appendix A.
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Klema, Valerie Jane. (2012). Toward a mechanistic understanding of cofactor biogenesis and catalysis in copper amine oxidases from Hansenula polymorpha.. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/131790.
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