Toward a Mechanistic Understanding of Cofactor Biogenesis and Catalysis in Copper Amine ...

199 Pages·2012·17.24 MB·English

Toward a Mechanistic Understanding of Cofactor Biogenesis and
Catalysis in Copper Amine Oxidases from Hansenula polymorpha

A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE
SCHOOL OF THE UNIVERSITY OF MINNESOTA

BY

VALERIE JANE KLEMA

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

ADVISER: CARRIE M. WILMOT

June, 2012
© Valerie Jane Klema 2012
Acknowledgements
Although only one name appears on the cover of this dissertation, a number of
people contributed to its completion and provided me with support that I will always
appreciate.
My sincerest gratitude goes to my adviser, Dr. Carrie Wilmot. Carrie has been a
true mentor, and has shown me incredible patience. I cannot imagine having completed
my graduate work with anyone else, and greatly appreciate the encouragement and
education that I have been given.
I would also like to acknowledge members of the Wilmot lab for all of their
assistance and ideas, as well as for creating a wonderful work environment. Many thanks
go to Dr. Lyndal Jensen, Corinne Solheid, Peder Cedervall, Dr. Brandon Goblirsch, Dr.
Erik Yukl, Dr. Teresa de la Mora, Dr. Bryan Johnson, and Dr. Chris Carrell.
My thanks go to members of the Lipscomb lab, particularly Dr. Melanie Rogers,
as well as Leah Randles and Dr. Kelaine Haas for advice and assistance with
experimental design. In addition, I am grateful to those who maintained the equipment
and computers which were necessary to my work, including Ed Hoeffner, Dr. Can
Ergenekan, and Nancy Rowe.
I have a wonderful group of friends who helped keep me sane through this
process, and appreciate their emotional support and camaraderie. Particular thanks go to
Molly Marshall, Kelaine Haas, and the members of HP.
Lastly, this dissertation would not have been possible without the support I
received from my parents, Martin and Rebecca Klema. No one has felt more love or
encouragement, of which I am more grateful than I can say.

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Dedication
This dissertation is dedicated to my parents, who have always believed in me. Words
cannot say how much I appreciate the unconditional love, support, and encouragement
you have offered throughout this process.

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Dissertation 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 O . Found in nearly every aerobic organism, CAOs take on a myriad of
2
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
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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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Table of Contents
List of Tables ................................................................................................................... vii
List of Figures ................................................................................................................. viii
List of Schemes ................................................................................................................. xi
List of Abbreviations ...................................................................................................... xii

Chapter 1: Introduction ....................................................................................................1
1.1 Background .................................................................................................................2
1.2 Physiological significance of CAOs ...........................................................................3
1.3 Post-translationally modified amino acid cofactors ....................................................6
1.4 Overall CAO fold ........................................................................................................9
1.5 Structure of the CAO active site ...............................................................................11
1.6 Biogenesis of TPQ ....................................................................................................13
1.7 Catalysis in CAOs .....................................................................................................23
1.8 CAO channels and access to the active site ..............................................................33
1.9 Research goals ..........................................................................................................38

Chapter 2: The precursor form of Hansenula polymorpha copper amine oxidase-1 in
complex with Cu(I) and Co(II) .......................................................................................41
2.1 Background ...............................................................................................................43
2.2 Methods.....................................................................................................................47
2.3 Results .......................................................................................................................51
2.4 Discussion .................................................................................................................62

Chapter 3: Structural and kinetic analysis of substrate specificity in two copper
amine oxidase paralogs from Hansenula polymorpha ..................................................71
3.1 Background ...............................................................................................................74
3.2 Methods.....................................................................................................................77
3.3 Results .......................................................................................................................79
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3.4 Discussion .................................................................................................................98
3.5 Conclusions .............................................................................................................105

Chapter 4: Understanding amine substrate specificity through the structural
analysis of Hansenula polymorpha copper amine oxidase-1 substrate complexes ...106
4.1 Background .............................................................................................................108
4.2 Methods...................................................................................................................112
4.3 Results .....................................................................................................................118
4.4 Discussion ...............................................................................................................139

Chapter 5: Ongoing works ............................................................................................148
5.1 Hansenula polymorpha copper amine oxidase mutants that alter substrate
specificity ...................................................................................................................149
5.2 Hansenula polymorpha copper amine oxidase-1 aniline derivatives .....................153
5.2.1 Methods................................................................................................................158
5.2.2 Preliminary results ...............................................................................................160

References Cited.............................................................................................................165

Appendix A: Chapter 2 supplementary methods .......................................................180

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List of Tables
1.1 TPQ-containing CAOs with available X-ray crystal structures .....................................9
1.2 λ of spectroscopic intermediates formed during CAO catalysis ............................ 28
max

2.1 k for biogenesis in HPAO-1 and AGAO supported by various metals ...................44
TPQ
2.2 X-ray data collection, processing, and refinement statistics for apoHPAO-1, Cu(I)-
apoHPAO-1, and Co(II)-apoHPAO-1 ............................................................................52

3.1 Comparison of k values in EDTA-dialyzed vs. non-dialyzed HPAO-2 samples ......82
cat
3.2 Steady state kinetic parameters for HPAO-1 and HPAO-2 .........................................84
3.3 X-ray crystallographic data collection, processing, and refinement statistics for
HPAO-2 ..........................................................................................................................87
3.4 Residue changes between HPAO-2 and HPAO-1 that likely impact accommodation
of substrate and catalytic intermediate structures ...........................................................95
3.5 Residue changes which define the O anteroom ........................................................103
2

4.1 X-ray diffraction, processing, and refinement statistics for methylamine-,
ethylamine-, and benzylamine-HPAO-1 complexes.....................................................118
4.2 Unit cell parameters for native HPAO-1 and substrate-HPAO-1 complexes. ...........119
4.3 Features in the substrate-HPAO-1 complex active sites. ...........................................126
4.4 Steady state kinetic parameters for the oxidation of methylamine, ethylamine, or
benzylamine by HPAO-1. .............................................................................................142

5.1 Steady state kinetic data for native HPAO-1, Y323C HPAO-1, native HPAO-2, and
C306Y HPAO-2 when reacted with benzylamine or methylamine ..............................151
5.2 X-ray crystallographic data collection, processing, and refinement statistics for
Y405pAF HPAO-1 .......................................................................................................161

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List of Figures
1.1 Quinone-containing cofactors formed by the post-translational modification of
tyrosine or tryptophan side chains ....................................................................................7
1.2 Structures of initially proposed CAO cofactors (A) PLP and (B) PQQ ........................8
1.3 Overall fold of HPAO-1 viewed along the molecular dyad axis .................................10
1.4 Diagram of the HPAO-1 active site with TPQ in its (A) “off-copper” or (B) “on-
copper” conformation .....................................................................................................13
1.5 Structurally characterized TPQ biogenesis intermediates in AGAO ...........................18
1.6 Amino acid residues in HPAO-1 which are involved in stabilizing biogenesis
intermediates ...................................................................................................................21
1.7 Solution UV/visible spectra showing the time course of the aerobic reconstitution of
apoHPAO-1 with Cu(II) .................................................................................................22
1.8 Structurally characterized intermediates formed during the CAO reductive half-
reaction ............................................................................................................................29
1.9 Structurally characterized intermediates formed during the CAO oxidative half-
reaction ............................................................................................................................32
1.10 Diagram of the amine substrate channel in different CAOs ......................................35
1.11 Overlay of xenon binding sites in xenon/CAO complexes ........................................37

2.1 UV/visible spectra showing the time course of the aerobic reconstitution of
apoHPAO-1 with Cu(II) at pH 7.0 ..................................................................................51
2.2 Arrangement of the domains and β-hairpin arms in apoHPAO-1 ...............................54
2.3 Stereoview of the active sites of (A) apoHPAO-1 (B) Cu(I)-apoHPAO-1 and (C)
Co(II)-apoHPAO-1 .........................................................................................................56
2.4 Stereoviews of newly modeled C-terminal residues in Cu(I)-apoHPAO-1. (A) Close-
up view of the new residues. (B) Newly modeled residues in Cu(I)-apoHPAO-1 within
the context of the HPAO-1 homodimer. (C) The second disulfide bond in Cu(I)-
apoHPAO-1 with 2F -F electron density ......................................................................59
o c
2.5 Stereoview of Co(II)-apoHPAO-1 active site residues with the 2F -F electron
o c
density map .....................................................................................................................60
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