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Chemistry and biochemistry of flavoenzymes.

Includes bibliographical references and indexes.

1. Flavoproteins. 2. Flavins. I. Muller, Franz,
1934-
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 PREFACE
Flavin was discovered about a century ago. Since its discovery, the field of flavin and
flavoproteins has developed into a mature science. Although a few flavoproteins had already
been discovered during the first three decades of the 20th century, a profound understanding
of the enzymic reaction mechanisms of flavoproteins became possible only after full de-
velopment of the chemical and physical properties of free flavin. This development was
initiated about 60 years ago and was mainly concerned with the chemical synthesis of the
flavin molecule. Research in the field of flavins and flavoproteins was discontinued during
the Second World War, but revived in the 1950s. The postwar period brought increased
progress in the chemistry of free flavin, which knowledge was instrumental in understanding
the biochemical properties of flavoproteins.
Flavoproteins are involved in a variety of biological reactions including electron transfer,
oxidation, dehydrogenation, and monooxygenation reactions. Many flavoproteins contain
flavin as the sole prosthetic group, but the reaction mechanisms can still be complex. Other
flavoproteins are composed of flavin and heme and non-heme iron, molybdenum and other
metal ions, or pteridin. Although they represent a minority, flavoproteins resulting from
covalent bondings of the prosthetic group to amino acid residues of protein have enhanced
the variety of flavoproteins.
Present knowledge in the field discussed in this work would not be possible without
advancements in biophysical equipment. New techniques have contributed a great deal to
the elucidation of primary, secondary, tertiary, and quarternary structures of flavoproteins.
In recent years the three-dimensional structures of a few flavoproteins have become available,
and it can be expected that this research will continue to advance. These data will provide
the basis for new research on flavoproteins — namely, cloning of genes encoding for
flavoproteins followed by site-directed mutagenesis of flavoproteins. This research is pres-
ently in its infancy, but is expected to develop and provide even deeper insight into the
enzymic mechanisms of flavoproteins and the biochemical principles governing the catalytic
action of particular flavoenzymes.
Given the wealth of data available on flavins and flavoproteins, it seems appropriate to
report on the progress of a century of research in the field. In the present work an attempt
has been made to summarize this knowledge as comprehensively as possible. Despite lim-
itations of space and unavailability of some material, this and succeeding volumes of the
Chemistry and Biochemistry of Flavoenzymes should be a valuable reference for researchers
in the field and those entering it as well as a source for lecturers in biochemistry, biophysics,
chemistry, pharmacology, toxicology, and medicine.
In conclusion I wish to thank all contributors for their cooperation and their efforts to
meet our deadlines, and I would also like to express personal appreciation to my wife, Rita,
and our daughters, Sandra and Kirsten, for their patience over many weekends in the
development of this series.

Franz Miiller
 THE EDITOR
Franz Miiller, Ph.D., is Head, Central Development Toxicology, Sandoz Agro Ltd.,
Basle, Switzerland, and Adjunct Professor of Biochemistry, University of Georgia, Athens,
Georgia.
Dr. Miiller obtained his training at the University of Basle, and received his M.S. degree
in 1961 and Ph.D. degree in 1964 from the Departments of Organic and Inorganic Chemistry.
After doing postdoctoral work at the Medical Nobel Institute, Department of Biochemistry,
Stockholm, Sweden, he was first a Research Associate in the Department of Biological
Chemistry at the University of Michigan in Ann Arbor, and later Instructor, and a recipient
of a Career Research Development Award from the National Institutes of Health. In 1971
he was appointed an Associate Professor of Biochemistry at the Agricultural University,
Wageningen, The Netherlands, and Research Professor in 1977, where he served as Chairman
of the Department of Biochemistry and chaired the university's Science Committee and the
Committee for Biotechnology. In 1987 he assumed his present position.
Dr. Miiller is a member of the Swiss Chemical Society, the Swiss Association of
Chemists, the Federation of European Biochemical Societies and the New York Academy
of Sciences.
He received the Venia Legendi (Habilitation) from the University of Constance, Federal
Republic of Germany, in 1970.
Dr. Miiller has been the recipient of research grants from the Netherlands Science
Foundation. He was also a member of the Committee for Natural Sciences of the grant
agency and chaired the Section for Protein Chemistry. He has published more than 200
papers. His current research interests are in biochemical transformation of pesticides.
 CONTRIBUTORS
Alessandro Aliverti, Ph.D. Eiji Itagaki, Ph.D.
Department of General Physiology and Associate Professor of Biochemistry
Biochemistry Department of Chemistry
University of Milan Kanazawa University
Milan, Italy Kanazawa, Japan

Robert G. Bartsch, Ph.D. Marilyn Schuman Jorns, Ph.D.

Research Professor Professor
Department of Biochemistry Department of Biological Chemistry
University of Arizona Hahnemann University
Tucson, Arizona Philadelphia, Pennsylvania

Christopher Bryant, Ph.D. Masayuki Katagiri, Ph.D.

Department of Chemistry Professor Emeritus
University of California, San Diego Department of Chemistry
La Jolla, California Kanazawa University
Kanazawa, Japan
Michael A. Cusanovich, Ph.D.
Professor Kalla Kvalnes-Krick, Ph.D.
Department of Biochemistry Postdoctoral Fellow
University of Arizona Department of Biochemistry and
Tucson, Arizona Biophysics
University of North Carolina
Karl Decker, Ph.D. Chapel Hill, North Carolina
Professor
Institute of Biochemistry Florence Lederer, Ph.D.
University of Freiburg Directeur de recherche
Freiburg, Germany Hospital Necker
Paris, France
Jan Drenth, Ph.D.
Professor Emeritus John Lee, Ph.D.
Laboratory for Chemical Physics Professor
University of Groningen Department of Biochemistry
Groningen, The Netherlands University of Georgia
Athens, Georgia
Sandro Ghisla, Ph.D.
Professor Vincent Massey, Ph.D.
Department of Biology Professor
University of Constance Department of Biological Chemistry
Constance, Germany University of Michigan
Ann Arbor, Michigan
Wim G. J. Hoi, Ph.D.
Professor Iain B. C. Matheson, Ph.D.
Laboratory of Chemical Physics Department of Biochemistry
University of Groningen University of Georgia
Groningen, The Netherlands Athens, Georgia
F. Scott Mathews, Ph.D. Lawrence L. Poulsen, Ph.D.
Professor Research Scientist Associate
Department of Cell Biology Department of Chemistry and
Washington University Medical School Biochemistry
St. Louis, Missouri University of Texas
Austin, Texas
William D. McElroy, Ph.D.
Professor Herman A. Schreuder, Ph.D.
Department of Chemistry Laboratory of Chemical Physics
University of California, San Diego University of Groningen
La Jolla, California Groningen, The Netherlands

Terrance E. Meyer, Ph.D. Helmut Simon, Ph.D.

Research Professor Professor
Department of Biochemistry Department of Organic Chemistry
University of Arizona Technical University of Munich
Tucson, Arizona Garching, Germany
Retsu Miura, Ph.D.
Thomas P. Singer, M.D., Ph.D.
Professor
Professor
Department of Chemistry
Department of Biochemistry and
Kansai Medical University
Biophysics
Osaka, Japan
University of California
San Francisco, California
Franz Miiller, Ph.D.
and
Head
Molecular Biology Division
Central Development Toxicology
Veterans Affairs Medical Center
Sandoz Agro, Ltd.
San Francisco, California
Basle, Switzerland
and
Adjunct Professor Daniel J. Steenkamp, Ph.D.
Department of Biochemistry Professor
University of Georgia Department of Chemical Pathology
Athens, Georgia University of Cape Town Medical School
Capetown, South Africa
Halina Y. Neujahr, Ph.D.
Professor Colin Thorpe, Ph.D.
Department of Biochemistry Professor of Biochemistry
Royal Institute of Technology Department of Chemistry and
Stockholm, Sweden Biochemistry
University of Delaware
Yasuki Nonaka, M.D., Ph.D. Newark, Delaware
Assistant Professor
Department of Biochemistry Willem J. H. van Berkel, Ph.D.
Osaka University Medical School Department of Biochemistry
Osaka, Japan Agricultural University
Wageningen, The Netherlands
Dennis J. O'Kane, Ph.D.
Assistant Biochemist Jan M. van der Laan, Ph.D.
Department of Biochemistry Laboratory of Chemical Physics
University of Georgia University of Groningen
Athens, Georgia Groningen, The Netherlands
Jacques Vervoort, Ph.D. Toshio Yamano, M.D., Ph.D.
Department of Biochemistry Professor Emeritus
Agricultural University Department of Biochemistry
Wageningen, The Netherlands Osaka University Medical School
Osaka, Japan

Giuliana Zanetti, Ph.D.

Professor of Biochemistry
Antonie J. W. G. Visser, Ph.D. Department of General Physiology and
Department of Biochemistry Biochemistry
Agricultural University University of Milan
Wageningen, The Netherlands Milan, Italy
 TABLE OF CONTENTS
Volume II

Chapter 1
Flavin-Dependent Monooxygenases with Special Reference to p-Hydroxybenzoate
Hydroxylase 1
Willem J. H. van Berkel and Franz Muller

Chapter 2
The Structure of/7-Hydroxybenzoate Hydroxylase 31
Herman A. Schreuder, Jan M. van der Laan, Wim G. J. Hoi, and Jan Drenth

Chapter 3
Phenol Hydroxylase 65
Halina Y. Neujahr

Chapter 4
The Multisubstrate FAD-Containing Monooxygenase 87
L, L. Poulsen

Chapter 5
A Steroid Ketone Monooxygenase from Cylindrocarpon radicicola 101
Masayuki Katagiri and Eiji Itagaki

Chapter 6
The Mechanism of Bacterial Bioluminescence 109
John Lee, Iain B. C. Matheson, Franz Muller, Dennis J. O'Kane, Jacques Vervoort,
and Antonie J. W. G. Visser

Chapter 7
Flavocytochrome b2 153
Florence Lederer

Chapter 8
L-Lactate Oxidase 243
Sandro Ghisla and Vincent Massey

Chapter 9
Nitroreductases 291
Christopher Bryant and William D. McElroy

Chapter 10
Ferredoxin: NADP+ Oxidoreductase 305
Giuliana Zanetti and Alessandro Aliverti

Chapter 11
Enoate Reductase 317
Helmut Simon
Chapter 12
NADPH-Adrenodoxin Oxidoreductase 329
Yasuki Nonaka, Retsu Miura, and Toshio Yamano

Chapter 13
Covalent Flavoproteins 343
Karl Decker

Chapter 14
Flavocytochrome c 377
Michael A. Cusanovich, Terrance E. Meyer, and Robert G. Bartsch

Chapter 15
The Biochemical Properties and Structure of Trimethylamine Dehydrogenase 395
Daniel J. Steenkamp and F. Scott Mathews

Chapter 16
Role of the Covalent and Noncovalent Flavins in Sarcosine Oxidase 425
Kalla Kvalnes-Krick and Marilyn Schuman Jorns

Chapter 17
Monoamine Oxidases 437
Thomas P. Singer

Chapter 18
Electron-Transferring Flavoproteins 471
Colin Thorpe

Index 487
 Volume II 1

Chapter 1

FLAVIN-DEPENDENT MONOOXYGENASES WITH SPECIAL

REFERENCE TO />-HYDROXYBENZOATE HYDROXYLASE
Willem J. H. van Berkel and Franz Muller

TABLE OF CONTENTS
I. Introduction 2

II. Internal Monooxygenases 2

III. External Monooxygenases 2

A. Overview 2
B. p-Hydroxybenzoate Hydroxylase 8
1. Enzyme Purification and Molecular Properties 8
2. Substrate Specificity 9
3. Interaction with Electron Donor 11
4. Enzyme Inhibition 12
5. On the Reaction Mechanism 13
6. Replacement of FAD by Modified Flavins 18
7. Chemical Modification Studies 19

IV. Conclusion 24

Acknowledgments 24

References 24
2 Chemistry and Biochemistry of Flavoenzymes

I. INTRODUCTION
Oxygenases are a class of proteins which catalyze the incorporation of one atom of
oxygen into the substrate molecule. The oxygen atom incorporated into the substrate is
exclusively derived from molecular oxygen (for an overview, see Reference 1). This was
unambiguously proven by using 18 O2 as a cosubstiate.
Oxygenases participate in many biological reactions such as biosynthesis and degradation
of a large variety of compounds, e.g., amino acids, lipids, porphyrins, vitamins, and hor-
mones. In recent years these enzymes received a great deal of attention because of their
involvement in the metabolic disposal of a variety of drugs and xenobiotics.
There are two classes of oxygenases. The dioxygenases catalyze the incorporation of
both atoms of dioxygen into the substrate. The monooxygenases, also referred to as "mixed
function oxygenases", catalyze the incorporation of a single atom of oxygen into the sub-
strate, while the other atom of dioxygen is reduced to water.
In this chapter we will be dealing with monooxygenases containing only flavin as a
prosthetic group. The flavoproteins in this class are conveniently divided into two categories:
(1) internal and (2) external monooxygenases. In the following material, the internal mo-
nooxygenases will be discussed briefly. The external monooxygenases, comprising a large
number of enzymes, will then be treated comprehensively.

II. INTERNAL MONOOXYGENASES

The internal flavin-dependent monooxygenases transform the substrate by hydroxylation
followed by decarboxylation, i.e., oxidative decarboxylation. The substrate serves also as
electron donor.
There are only a few internal monooxygenases known. Some properties of these enzymes
are presented in Table 1. One of these enzymes, lactate monooxygenase,2'3 will be discussed
as a representative of this class of enzymes in more detail in Chapters 7 and 8.
Arginine monooxygenase converts L-arginine into 7-guanidobutyramide. The substrate
specificity of the enzyme is fairly rigid.4 The pH optimum of the activity is in the region
of pH 9, and the enzyme exhibits a sigmoidal substrate saturation curve.4*5 The partially
purified enzyme is rather unstable in the presence of dioxygen. The enzyme can however
be stabilized to a certain degree by storage in a nitrogen atmosphere or by addition of
reducing agents like dithionite or thioglycolate.
Lysine monooxygenase converts L-lysine into 8-aminovaleramide. The enzyme has been
obtained in a crystalline state.6"8 When chemically reduced, the semiquinone of the enzyme
is formed and dependent on the pH the neutral or anionic radical is obtained. Only a few
flavoproteins show this behavior. The enzyme exhibits also other unusual properties. The
substrate shows sigmoidal saturation curves, as arginine monooxygenase, and the reaction
with lysine does not go to completion. The percent completion of the reaction depends on
the concentration of the substrate.9 The substrate serves also as an effector of the enzymatic
reaction.

III. EXTERNAL MONOOXYGENASES

A. OVERVIEW
The external flavoprotein monooxygenases are members of a class of inducible enzymes
catalyzing the insertion of one atom of molecular oxygen into the substrate using pyridine
nucleotides as external electron donors. The overall reaction is
 Volume II 3

TABLE 1
Some Properties of Internal Monooxygenases

Origin Enzyme code Ref.

Reaction catalyzed Molecular mass

(1) Lactate 2-monooxygenase (FMN)a [EC1.13.12.4]

((S)-Lactate: oxygen 2-oxidore-
ductase (decarboxylating))
R-CHOH-COOH + O2 R-COOH + C02 + H20
Mycobacterium phlei 55—56 kDab 2, 3
Mycobacterium smegmatis 300 kDa (hexamer) 3

(2) Arginine 2-monooxygenase (FAD)a [EC 1.13.12.1]

(L-arginine: oxygen 2-oxidore-
ductase (decarboxylating))
arginine + O2 y-guanidobutyramide + CO2 + H2O
Streptomyces griseus c 4
Acetomyces species c
4

(3) Lysine 2-monooxygenase (FAD)a [EC 1.13.12.2]

(L-Iysine: oxygen 2-oxidore-
ductase (decarboxylating))
L-lysine + O2 6-aminovaleramide
Pseudomonas fluorescens 246 kDa (tetramer) 6—8

a
Prosthetic group.
b
Minimum molecular mass.
c
Molecular mass not known.

Until now about 25 external flavin-dependent monooxygenases have been isolated and
(partially) characterized (Table 2). The enzymes listed in Table 2 show considerable dif-
ferences in both molecular mass and subunit composition. Except for the a2(32 tetrameric
2,4-dichlorophenol hydroxylase from Alcaligenes eutrophus,35 all multimeric, external flavin-
dependent monooxygenases are built up by identical subunits, each containing noncovalently
bound flavin adenine dinucleotide (FAD) as a prosthetic group. With some enzymes FAD
is partly lost during purification. Only in the case of cyclohexanone 1,2-monooxygenase
from a cyclohexane-degrading Xanthobacter species, it was reported that protein-bound
riboflavin 5'-phosphate (FMN) replaces FAD as electron acceptor.62
Most FAD-dependent monooxygenases have been isolated from aerobic prokaryotes able
to degrade aromatic or cyclic ketone compounds.67'70 These bacteria (especially soil pseu-
domonads) can grow on different substrates as sole carbon sources thereby inducing the
corresponding flavoprotein monooxygenases. Through the initial action of these enzymes,
the resulting products are readily subject to further catabolism, allowing microbes to grow.
In this way breakdown products of lignin as well as many pollutants and toxicants (e.g.,
pesticides) can be metabolized. It should be noted that nonactivated aromatic compounds
like benzene or benzoate cannot be hydroxylated by the FAD-dependent monooxygenases.
Metabolism of this type of compounds requires the initial action of both flavin and nonheme
iron-containing enzyme systems showing a more potent oxygenating capacity.70
Many FAD-dependent monooxygenases prefer either NADH or NADPH as a reductant,
though some enzymes can utilize both pyridine nucleotides with different efficiency in the
catalytic turnover (Table 2). All FAD-dependent monooxygenases studied so far show pref-
erence for abstraction of the pro-R hydrogen of the C-4 prochiral center of the dihydroni-
cotinamide moiety of the pyridine nucleotide.71*72
The other (no iron-containing) flavin-dependent monooxygenases have been reported to
receive their reducing equivalents in a different way. First, luciferase from bioluminiscent
4 Chemistry and Biochemistry of Flavoenzymes

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