Structure, function and evolution of flavin/deazaflavin dependent oxidoreductases

199 Pages·2016·29.79 MB·English

by  Hafna Ahmed

Structure, function and evolution of
flavin/deazaflavin dependent oxidoreductases
(FDORs) in mycobacteria
A thesis submitted for the degree of Doctor of Philosophy of
the Australian National University

Fathimath Hafna Ahmed
April 2016

© Copyright by Fathimath Hafna Ahmed 2016
All Rights Reserved

Declaration
This thesis presents my original work carried out in the laboratory of Associate
Professor Colin Jackson unless otherwise stated. Author contributions have been clearly
stated preceding the 5 peer-reviewed publications that are presented as individual
chapters. This includes the published review in Chapter 2 co-first authored with Dr.
Chris Greening, the published article in Chapter 4 primarily carried out by Elaaf
Mohamed and the published article in Chapter 6 co-first authored with Blair Ney. The
key findings for this thesis from each publication are summarised following the
manuscripts. The work I have performed has not been previously submitted for another
degree or diploma in any university or tertiary institution.

F. Hafna Ahmed
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Acknowledgements
Alhamdulillah for all the unlikely opportunities that got me here.
I want to thank Assoc. Prof. Colin Jackson for the opportunity to work with him on this
challenging project and for looking out for me during these past 4 years. I may literally
be stuck on an island otherwise so don’t think I need to elaborate more on how grateful
I am to him.
I also would like to thank our collaborator Dr. Chris Greening at CSIRO who helped me
with organising and writing up the manuscripts presented in this thesis. Thank you to
Dr. Paul Carr for being extremely patient in teaching me crystallography and Dr. Livnat
Afriat-Jurnou for helping me make sense of this project during my first year. I also want
to acknowledge Dr. Matt Taylor from CSIRO who gave us a lot of the FDOR gene
constructs and the rest of the FDOR team in the lab, especially Brendon Lee who
contributed very significantly to finishing up my first publication.
And of course my husband Elaaf Mohamed who did the physical chemistry part of this
project. Even though it wasn’t ideal, I can’t think of a better person to share a PhD
project with. After this, raising our child currently residing in my belly should be no
problem…right?
Thank you to all the past and present members of the Jackson lab, your support and the
ridiculous things we occasionally got up to made these past years some of the most
memorable of my life. I also would like to thank the other wonderful people at the RSC,
especially the support staff and the members of the Coote and O’Mara groups for
regularly listening to my whining when I visit Elaaf. And a big thank you to my friends
who got frequently neglected due to my obsession with work and the Maldivians in
Canberra for being my family away from home.
I wouldn’t be here if it wasn’t for my family – my aunts, uncles, cousins and in-laws –
who have always encouraged my interest in science and are my biggest supporters. And
of course my sisters, Hasna for her regular reminders of how dumb I really am and
Hilya for being my motivation.
Lastly I want to thank my Mom and Dad, there is nothing more inspiring than their hard
work and dedication to make a success out of what life dealt them, so this is their
achievement as much as mine.

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To my baby sister Hilya.
I wished every day that I didn’t have to miss your childhood for this,
may it inspire you to reach for the stars and persevere.

And to Aya.
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Abstract
Mycobacterium tuberculosis, the causative agent of Tuberculosis (TB), is notorious for
its ability to persist during infection and evade the host immune response, although the
cellular mechanisms behind this are not yet fully understood. Like other mycobacteria,
M. tuberculosis produces the cofactor F found in Actinobacteria and Archaea, which
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is important, through unknown mechanisms, for its survival during oxidative stress and
in the reactivation of latent infection. This project presents the characterisation of the
largest F utilizing protein family in mycobacteria known as the flavin/deazaflavin
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dependent oxidoreductases (FDORs), forming the basis for identifying potential
functional roles of these enzymes that might contribute to mycobacterial infection and
persistence.
While most FDORs in mycobacteria utilise F as a cofactor, this family also includes
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proteins with other cofactor specificities, including proteins that utilise FMN, FAD and
heme. Five novel FDOR structures are presented, which, along with previously
available structures, allowed the identification of conserved motifs to differentiate
between FDORs with different cofactor specificities. Comparisons between the
structures also showed that FDORs have relatively conserved cofactor-binding regions,
while the substrate binding pockets are extensively modified for functional adaptation.
Their cofactor preference, sequence similarity and structures allowed the classification
of the FDORs into functional groups, including the previously identified F H -
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dependent quinone reductases that also activate 4-nitroimidazole pro-drugs approved for
treating multi-drug resistant M. tuberculosis infection. For the first time, mycobacterial
heme oxygenases belonging to this family were also found, along with novel FAD
binding proteins that could be involved in the hypoxia response that triggers
mycobacterial dormancy. Furthermore, in silico substrate docking led to the
identification of novel F H -dependent fatty acid saturases and F H -dependent
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biliverdin reductases (F-BVRs) within the FDORs.
Detailed characterisation of the F-BVR Rv2074 from M. tuberculosis showed that its
homologues are present in pathogenic and commensal mycobacteria and that it reduces
biliverdin-IXα (the principle isomer produced by human macrophages) to bilirubin-IXα.
Bilirubin is a potent antioxidant that could contribute to M. tuberculosis surviving
oxidative stress encountered inside macrophages. Rv2074 reduces biliverdin by a
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mechanism similar to the nicotinamide-dependent reactions in the mammalian
biliverdin reductases as inferred using the structure of the Rv2074:F complex with
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biliverdin modelled into the active site. Proton donation to a pyrrole nitrogen occurs
first from a hydroxonium ion stabilised by an arginine residue, which is consistent with
the requirement for an alternate proton donor since F H appears to be stabilised in its
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deprotonated state when bound to FDORs. The resulting cationic intermediate
undergoes hydride transfer with F H , completing bilirubin formation.
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Lastly, F production was found to be more widespread than in just Actinobacteria and
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Archaea, with data confirming F production in some Proteobacteria and Chloroflexi,
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which also encode the proteins required for its biosynthesis. FDORs are also present in
these organisms, implying their capability of utilising this rare cofactor as well.
Overall, the work described in this thesis highlights the diversity of the FDORs and has
identified functional roles that could contribute to M. tuberculosis pathogenesis and
persistence by enhancing survival during oxidative stress.

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Contents

Declaration ........................................................................................................................ 1
Acknowledgements ........................................................................................................... 3
Abstract ............................................................................................................................. 5
Contents ............................................................................................................................ 7
Abbreviations .................................................................................................................... 9
Chapter 1 – Thesis Outline .............................................................................................. 11
1.1 Introduction ........................................................................................................... 12
1.2 Research objectives ............................................................................................... 13
1.3 Research outline .................................................................................................... 14
1.4 Experimental methods ........................................................................................... 15
1.5 Publications ........................................................................................................... 15
1.6 Protein structure depositions ................................................................................. 16
Chapter 2 – Literature review ......................................................................................... 17
2.1 Summary ............................................................................................................... 18
2.2 Published Article ................................................................................................... 20
Chapter 3 – Sequence, structure and function of FDORs in mycobacteria .................. 65
3.1 Introduction ........................................................................................................... 66
3.2 Published article .................................................................................................... 67
3.3 Structure of the FAD binding protein MSMEG_5243 .......................................... 95
3.4 Implications, key findings and future directions ................................................... 97
Chapter 4 – Protonation state of F H when bound to FDORs ................................... 99
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4.1 Introduction ......................................................................................................... 100
4.2 Published article .................................................................................................. 101
4.3 Implications, key findings and future directions ................................................. 114

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Chapter 5 – Structure and mechanism of the F H -dependent biliverdin reductase
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Rv2074 from Mycobacterium tuberculosis .................................................................. 115
5.1 Introduction ......................................................................................................... 116
5.2 Published article .................................................................................................. 117
5.3 Implications, key findings and future directions ................................................. 136
Chapter 6 – Abundance and distribution of F producing organisms ...................... 139
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6.1 Introduction ......................................................................................................... 140
6.2 Published article .................................................................................................. 141
6.3 Implications, key findings and future directions ................................................. 164
Chapter 7 – General Discussion ................................................................................... 165
7.1 Overview ............................................................................................................. 166
7.2 FDORs in mycobacterial persistence and pathogenesis ..................................... 166
7.3 Catalytic mechanisms of F H -dependent FDORs ........................................... 168
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7.4 FDOR evolution .................................................................................................. 169
7.5 F and FDORs in other bacteria ....................................................................... 171
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7.6 Future directions ................................................................................................. 172
7.6 Conclusions ......................................................................................................... 172
References ..................................................................................................................... 173
Appendix ....................................................................................................................... 185
Appendix I: General background on protein X-ray crystallography ........................ 186
Appendix II: General background to phylogenetics and SSNs................................. 191

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