Table Of ContentTHE SHIKIMATE PATHWAY IN NEISSERIA GONORRHOEAE
By
PREM S. SUBRAMANIAM
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1993
To the memories, dreams and aspirations of my mother and
father.
.
ACKNOWLEDGEMENTS
First and foremost, I would like to express my deepest
gratitude to and highest admiration for my guide and mentor,
Dr. Roy A. Jensen. His compassionate soul, brilliant mind,
reassuring philosophies, perseverence and patience have
allowed me to complete this journey. His infectious thirst
forscientific knowledge has, forever, transformedmyapproach
to scientific inquiry. His continued financial support is
most gratefully acknowledged.
I would like to thank Dr. John E. Gander, Dr. James F.
Preston, Dr. K. T. Shanmugam, and Dr. Charles M. Allen for
agreeing to serve onmygraduate committee, for their generous
amounts of time, and for their insights, guidance and advice
at the various stages in the completion of this dissertation.
A special thanks goes to Carol Bonner, for her support,
concern and friendship through many uncertain times.
I wish also to express my thanks to Dr. Randy Fischer for
his generosity, in time and advice; his support, expertise and
friendship have been invaluable
Few words can express my gratitude to my brother and
sister-in-law for their unfailing support, encouragement,
succour, generosityand selflessness to ensure the fruitionof
this dissertation.
iii
.
I am indebted to my friends Mridul, Radhika, Shankar,
Rajul, Mohan and Meena for their unimaginable levels of
support, encouragement and companionship that have helped me
reach this goal.
I would like to acknowledge the congeniality and
assistance of all my lab-mates past and present: Dr. Paul
Morris, Dr. Raj Bhatnagar and Dr. Suhail Ahmad for their
guidance and assistance during my initial stages; Dr. Ron Lou
Doong for his technical intensity; Dr. Genshi Zhao for his
companionship and support; Dr. Tianhui Xia forhis imaginative
insights; Jian Song (for "talk radio"!); and Wei-Wei (also
known as Wei Gu!) for making my passage pleasant and
memorable. A special thanks goes to Mrs. Premila Rao for her
concerned friendship andsociability (andhertireless efforts
on prephenate and arogenate
!)
Finally, but not the least, avery special thanks to goes
my fiancee, Anuradha, for her love, patience, encouragement
and understanding, which I have been so lucky to have grace
the latter portions of my quest.
iv
TABLE OF CONTENTS
ACKNOWLEDGEMENTS iii
LIST OF TABLES vii
LIST OF FIGURES viii
ABSTRACT X
CHAPTERS
1 INTRODUCTION 1
The Shikimate Pathway for Biosynthesis of
Aromatic Amino Acids 4
The Aromatic Biosynthetic Pathway in
N.gonorrhoeae 7
Key Initial Results Fundamental to
Formulation of Objectives 10
Project Objectives 12
2 PROTEIN-PROTEIN INTERACTIONS WITHIN THE EARLY
PATHWAY OF AROMATIC BIOSYNTHESIS IN NEISSERIA
GONORRHOEAE 14
Introduction 14
MateriCahlesmicaanldsMeatnhdodBsiochemicals .... 1166
Bacterial Strains, Media and Growth
Conditions 17
Chromatography 19
Enzyme Assays 21
Results and Discussion 23
Are the Two DAHP Synthases the
The PrMoodnuocmteroifc theFoSramme Goefne? DA.HP. 34
Synthase 37
Gel Filtration of Crude Extracts
Point to the Association of the
Activities of DAHP Synthase and
Shikimate Dehydrogenase ... 42
The Monomeric Nature of Shikimate
Dehydrogenase 45
v
Purification of Shikimate
Dehydrogenase 48
Analysis of the Purified
Preparation 52
Indirect Evidence for the
Involvement of Dehydroquinate
Synthase in the Complex ... 60
Regulation of the Aromatic Pathway
at the Level of DAHP Synthase
The aDnSd-PShheikiimsaotzeymDeehydirnogetnhaese /3-. 65
subdivision of Purple
Bacteria 68
3 THE OVERLAPPING SUBSTRATE-UTILIZATION
CAPABILITIES OF DAHP SYNTHASE AND KDOP
SYNTHASE 77
Introduction 77
Results and Discussion 79
RelaxeKdDOSPubSsytnrtahtaeseSpReecaicftiicointy.o.f.th.e 79
The Stereochemistryof the Resultant
SubstrCa7tPer-oadmubcitguity and Reversal of 84
Stereospecificity as More
Prevalent Characteristics of
DAHP- and KDOP Synthases. . 97
4 CONCLUSIONS 100
RPeecrosnpsecttiitvuetsionandofFututrhee DMiurlecttiifounnscti.on.al. 103
Complex 103
Are the Two Forms of DS-Phe Prevalent in
Superfamily A a Reflection of the
Above Complex? 105
REFERENCES 106
BIOGRAPHICAL SKETCH Ill
vi
LIST OF TABLES
Table page
2-1 List of N. gonorrhoeae strains used 18
2-2 Results of the purification protocol for shikimate
dehydrogenase 55
2-3 Distribution of DAHP synthase isozymes within
organisms of the /^-subdivision 75
vii
LIST OF FIGURES
Figure page
2-1 Elution profiles of DAHP synthase and shikimate
dehydrogenase from hydroxylapatite chromatography
of N. gonorrhoeae strain HGH 154 25
2-2 Inhibition of DAHP synthase by L-phenylalanine 27
2-3 Elution profile for DAHP synthase from
hydroxylapatite chromatography of crude extracts of
VHC 3102 30
2-4 Hydroxylapatite chromatography of crude extracts of
strain 2013 grown in the presence of phenylalanine,
tyrosine and tryptophan 33
2-5 Hydroxylapatite chromatography of crude extracts of
strain 2013-7 36
2-6 DEAE-cellulose chromatography of crude extracts of
strain 2013 39
2-7 Gel-filtration of DAHP synthase 41
2-8 Gel-filtration of a crude extract of strain 2013 on
a Bio-gel A-0.5m column 44
2-9 Gel-filtration of a crude extract from strain 2013
on a Bio-gel A-0.5m column 46
2-10 The monomeric form of shikimate dehydrogenase 50
2-11 Purification of shikimate dehydrogenase 54
2-12 Polyacrylamide-gel-electrophoresis analysis of the
purified preparation of shikimate dehydrogenase 57
2-13 DEAE-cellulose chromatography of crude extracts of
strain 2013 62
2-14 Affinity chromatography on 2'-5'-ADP agarose of the
enzymes eluted from DEAE cellulose as depicted in
viii
Fig. 2-13 64
2-15 Hydroxy1apatite chromatography of crude extracts of
strain 2013 66
2-16 Chromatography of strain 2013-8, a pFP-resistant
mutant 69
2-17 Kinetics of inhibition of SDH by chorismate 72
2-18 Activity profiles of enzymes from P. testosteroni
ATCC 17409 on DEAE-cellulose 74
3-1 Chromatography of crude extracts of Neisseria
gonorrhoeae VHC 3012 on hydroxylapatite 81
3-2 The relative ability of KDOP synthase to use E4P
and A5P at fixed concentration of PEP 83
3-3 Kinetics of periodate oxidation of the enzymatic
products of KDOP synthase and DAHP synthase
reactions with E4P and PEP 88
3-4 The reaction of C -X with dehydroquinate synthase 90
7
3-5 Chemical structures of DAHP, KDOP and DRHP 93
3-6 Representation of the mechanism of formation of
DAHP by DAHP synthase, KDOP by KDOP synthase and
DRHP by KDOP synthase 95
ix
Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
THE SHIKIMATE PATHWAY IN NEISSERIA GONORRHOEAE
By
Prem S. Subramaniam
December, 1993
Chairperson: Roy A. Jensen
Major Department: Microbiology and Cell Science
Neisseria gonorrhoeae was shown to possess a dissociable
complex comprising of a phenylalanine-inhibitable isozyme of
3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DS-Phe)
and shikimate dehydrogenase (SDH). Both of the enzymes were
active as monomers. DS-Phe activity was found to be
partitioned between associated (DS-PheL) and dissociated (DS-
Phe7) forms. A Mr of ~ 35,000 was estimated for the monomeric
form of DS-Phe by gel-filtration. DS-Phe was found as a dimer
(~70,000 daltons) in the presence of one of its substrates,
phosphoenolpyruvate (0.75 mM). AMr of ~ 27,000 was estimated
for the monomeric form of SDH by gel-filtration and
polyacrylamide gel-electrophoresis Gel-filtration analysis
.
of crude extracts showed that SDH and DS-Phe existed as
components of a larger unit of Mrbetween 150,000 and 200,000.
"Natural" aromatic amino acid auxotrophs of N. gonorrhoeae
were found to be lacking the activity of shikimate
x
