Báo cáo khoa học: Flavogenomics – a genomic and structural view of flavin-dependent proteins potx

The number of genes encoding flavin-dependent proteins varies greatly in the genomes analyzed, and covers a range from 0.1% to 3.5% of the predicted genes.. To verify the dependence of a

Flavogenomics – a genomic and structural view of

flavin-dependent proteins

Peter Macheroux1,2, Barbara Kappes3and Steven E Ealick2

1 Institute of Biochemistry, Graz University of Technology, Austria

2 Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA

3 Department of Parasitology, University Hospital Heidelberg, Germany

Keywords

enzymes; flavin adenine dinucleotide (FAD);

flavin mononucleotide (FMN); genomic

distribution; oxidoreductases; redundancy;

structures

Correspondence

P Macheroux, Institute of Biochemistry,

Graz University of Technology, Petersgasse

12 ⁄ II, A-8010 Graz, Austria

Fax: +43 316 873 6952

Tel: +43 316 873 6450

E-mail: peter.macheroux@tugraz.at

(Received 17 March 2011, revised 11 May

2011, accepted 31 May 2011)

doi:10.1111/j.1742-4658.2011.08202.x

Riboflavin (vitamin B2) serves as the precursor for FMN and FAD in almost all organisms that utilize the redox-active isoalloxazine ring system

as a coenzyme in enzymatic reactions The role of flavin, however, is not limited to redox processes, as 10% of flavin-dependent enzymes catalyze nonredox reactions Moreover, the flavin cofactor is also widely used as a signaling and sensing molecule in biological processes such as phototropism and nitrogen fixation Here, we present a study of 374 flavin-dependent proteins analyzed with regard to their function, structure and distribution among 22 archaeal, eubacterial, protozoan and eukaryotic genomes More than 90% of flavin-dependent enzymes are oxidoreductases, and the remaining enzymes are classified as transferases (4.3%), lyases (2.9%), isomerases (1.4%) and ligases (0.4%) The majority of enzymes utilize FAD (75%) rather than FMN (25%), and bind the cofactor noncovalently (90%) High-resolution structures are available for about half of the flavo-proteins FAD-containing proteins predominantly bind the cofactor in a Rossmann fold ( 50%), whereas FMN-containing proteins preferably adopt a (ba)8-(TIM)-barrel-like or flavodoxin-like fold The number of genes encoding flavin-dependent proteins varies greatly in the genomes analyzed, and covers a range from 0.1% to 3.5% of the predicted genes

It appears that some species depend heavily on flavin-dependent oxidore-ductases for degradation or biosynthesis, whereas others have minimized their flavoprotein arsenal An understanding of ‘flavin-intensive’ lifestyles, such as in the human pathogen Mycobacterium tuberculosis, may result in valuable new intervention strategies that target either riboflavin biosynthe-sis or uptake

Introduction

Biological cofactors are generally employed by enzymes

to enable a wide and diverse range of biochemical

transformations necessary for all aspects of life Some

of these cofactors, such as vitamin B12 and vitamin H

(biotin), catalyze a small but nevertheless important set

of biochemical reactions Other cofactors, on the other

hand, perform very different chemical tasks, and compete for the title of master of versatility, with vitamin B2 (riboflavin)-derived, vitamin B6-derived (e.g pyridoxine and pyridoxamine) cofactors and cytochrome P450 being the most serious contenders The yellow vitamin B2, or riboflavin, is synthesized by

Abbreviations

PDB, Protein Data Bank; RI, redundancy index.

many bacteria and plants [1,2], and then converted to

FMN and FAD (for structures see Fig 1) by riboflavin

kinase (which catalyzes the phosphorylation of the

ribityl side chain attached to N10 of the isoalloxazine

ring system) and further adenylated by FAD-synthetase

in two ATP-dependent reactions [3–5] These two

modified forms of riboflavin occur exclusively in

flavin-dependent enzymes The biochemical utility of FMN

and FAD is based on their redox-active isoalloxazine

ring system, which is capable of one-electron and

two-electron transfer reactions and, most importantly, of

dioxygen activation [6] Generations of enzymologists

have marvelled about the astonishing diversity of

flavin-dependent reactions, encompassing

dehydrogena-tion [7], oxidadehydrogena-tion [8–10], monooxygenadehydrogena-tion [11–13],

halogenation [14–16], and reduction (e.g of disulfides

and various types of double bond) [17], as well as their

utility in biological sensing processes (e.g light and

redox status) [18–25] Not surprisingly, this area has

been the subject of numerous review articles that have

attempted to fathom and rationalize the capabilities of

the flavin cofactor [26–32] The complexity of

flavin-catalyzed reactions is further increased when they join

forces with other redox-active cofactors, such as iron–

sulfur clusters ([2Fe–2S], [3Fe–4S] and⁄ or [4Fe–4S])

[33–35], heme [36], molybdopterin [37], or thiamine

diphosphate [38]

Since the discovery of the first flavin-containing

enzyme by Otto Warburg in the 1930s [39], the number

of ‘yellow’ enzymes has steadily increased, and there

has been a sharp rise in the last 20–30 years, owing to

the rapid progress in molecular cloning and full

genome sequencing More recently, structural genomics

has led to the structural characterization of many more

and hitherto unknown flavoproteins To gain an

over-view of flavoproteins, their genomic distribution, and

their structural topologies, we have assembled a list of

flavoproteins and searched for the encoding sequences

in a selection of genomes In addition, structural infor-mation on flavoproteins in the Protein Data Bank (PDB) was analyzed in order to define the flavin-bind-ing pocket accordflavin-bind-ing to the PFAM classification scheme [40]

Nature’s flavoprotein arsenal

The list of flavin-dependent proteins was assembled by using, mainly, three on-line databases First, the enzyme database BRENDA (http://www.brenda-enzy-mes.org/) was searched for FMN-dependent and FAD-dependent enzymes to compile a preliminary list This initial list contained many false positives and also missed several flavin-dependent enzymes, as well as flavoproteins with no catalytic or no known catalytic function (e.g flavin storage proteins) To verify the dependence of a protein on flavin, the primary litera-ture was consulted, and a complementary search for classified enzymes in the Enzyme Structures Database (http://www.ebi.ac.uk/thornton-srv/databases/enzymes/) and the PDB (http://www.pdb.org/pdb/home/home.do) was conducted to link the list of flavoproteins to the available structural information

The current list of flavoproteins contains 276 fully classified enzymes and 98 entries for enzymes with no

or incomplete classification as well as flavoproteins without a demonstrated enzymatic activity (cofactor storage, electron transfer, repressor and response proteins; 17 entries) As could be expected for a redox-active cofactor, the majority of flavoenzymes are found

in enzyme class 1: oxidoreductases account for 91% (251 entries), whereas transferases, lyases, isomerases and ligases contribute only 4.3% (12 entries), 2.9% (eight entries), 1.4% (four entries), and 0.4% (one entry) (Fig 2A) Within the class of oxidoreductases,

N

N

NH N

CH 2

O O

CH HC CH

OH OH OH

O P O P O

O O O

O

O N

N N

N

NH 2

OH OH

1 2 3 4 5 6 7 8 9 10

N

N

NH N

O O

H H

Isoalloxazine ring

Riboflavin

FAD FMN

2e / H

H 2 C

H 2 C

Fig 1 Structure of riboflavin, FMN, and FAD The redox-active isoalloxazine ring is shown in its oxidized and two-electron reduced state (red and blue) The numbering scheme for the isoalloxazine ring is indi-cated in the oxidized structure on the left.

the three largest subgroups are enzymes in EC 1.1.4

(61 entries for monooxygenases⁄ hydroxylases), EC 1.1

(38 entries for enzymes oxidizing a CH–OH group),

and EC 1.1.3 (30 entries for enzymes oxidizing a

CH–CH group) (Fig 2B)

FAD is clearly more common as a cofactor than

FMN, with 289 proteins depending on FAD (75%)

and 98 on FMN (25%) (note: entries where cofactor

utilization is unclear were not considered; see

Table S1) Riboflavin is not used in any enzymes

(except for riboflavin kinase⁄ FAD synthetase as a

sub-strate), but appears to be the preferred storage form of

the cofactor in some organisms (e.g riboflavin-binding

protein in chicken eggs and dodecin in archaeons

[41,42]) In addition, organisms (e.g mammals) lacking

vitamin B2 biosynthesis employ riboflavin-specific

transporters to sequester it from dietary sources by

facilitated diffusion [43]

In the majority of enzymes, the cofactor is

noncova-lently bound in the active site Covalent attachment of

the flavin cofactor has been confirmed in 40 cases (see

Table S2), corresponding to  10.8% of all

flavopro-teins listed in Table S1 Apparently, covalent

attach-ment of FMN (five entries) occurs rarely as compared

with that of FAD (35 entries) Different types of

cova-lent attachment have been found for FMN It is linked

either to the 8a-position (via N3 of a histidine) or to

the 6-position (via the thiol group of a cysteine) of the

isoalloxazine ring [44], or, in one case, it is bicovalently

linked to N1 of a histidine and the thiol group of a

cysteine [45] Only recently, a novel attachment of

FMN to redox-driven ion pumps (RnfG and RnfD)

via an ester linkage between the hydroxyl group of a

threonine and the ribitylphosphate side chain of the

cofactor was discovered [46] On the other hand,

cova-lent linkage of FAD always occurs via the 8a-position,

to either the N1 or N3 of a histidine, a cysteine thiol,

a tyrosine hydroxyl, or an aspartate carboxyl group (Table S2) [44,47] In five enzymes, FAD is

bicovalent-ly attached via the 8a-position and 6-position of the isoalloxazine ring system [48] Bicovalent attachment was first discovered only 5 years ago, but appears to

be more common than monocovalent attachment to the 8a-position via cysteine, tyrosine, or aspartate [49,50]

Flavoprotein structures

The first structure of a flavin-dependent protein was reported in 1972 for a bacterial flavodoxin [51,52] Sev-eral years later, the structures of the FAD-dependent enzymes glutathione reductase (EC 1.8.1.7) and 4-hy-droxybenzoate 3-monooxygenase (p-hy4-hy-droxybenzoate hydroxylase; EC 1.14.13.2) were described [53,54] Since that time, the numbers of deposited structures have risen to 646 and 1179 structures of FMN-depen-dent and FAD-depenFMN-depen-dent proteins, respectively (as of

31 December 2010), and this has been paralleled by efforts to relate the structures of flavoproteins to their functions [55–58] The structure of flavodoxin, a small electron transfer protein that uses FMN as a cofactor,

is not only the first but also by far the most frequently solved structure of all flavin-dependent proteins (> 120 entries in the PDB)

Currently, structures are available for 55 FMN-uti-lizing and 141 FAD-utiFMN-uti-lizing flavoproteins, accounting for  52% of all flavoproteins listed in Table S1 Overall, a total of 23 structural clans (according to the PFAM classification [40]) is represented by flavin-dependent proteins, and the structural topologies are therefore quite diverse in comparison with other cofactor-dependent enzyme families; for example, all pyridoxal 5¢-phosphate-dependent enzymes adopt one

of five different structural topologies [59]

6 (0.4%)

5 (1.4%)

4 (2.9%)

2 (4.3%)

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.14

1.16

1.17 1.18

1.21

1.13

1.12

1.11

1.10

1 (91%)

Fig 2 Pie chart of flavoproteins found in various enzyme classes: yellow, class 1 (oxidoreductases); orange, class 2 (transferases); red, class 4 (lyases); blue, class 5 (isomerases); and green, class 6 (ligases) This chart was generated by using the fully classified flavoenzymes (a total of 276) from Table S1.

As can be seen from Fig 3, FMN and FAD binding

are vastly different with respect to the topology of the

binding pocket, indicating that the adenosine moiety

strongly affects the mode of cofactor binding The

pre-ferred structure for FMN binding is the classical

(b⁄ a)8-barrel (clan TIM_barrel), with 16 entries, and

the flavodoxin-like fold (clan Flavoprotein), with 12

entries Together, these two clans account for more

than half of the currently known FMN-dependent

structural types Graphical representations of these

two most common topologies in FMN-dependent

pro-teins are shown in Fig 4A,B Within the clan

TIM_barrel, five families are found in FMN-dependent

enzymes: FMN_dh (six entries), Oxidored_FMN (five

entries), and DHO_dh, Glu_synthase and NPD (one

entry for each family) In the clan Flavoprotein, nine

proteins adopt a Flavodoxin_1, two an FMN_red and

one a recently discovered Flavodoxin_NrdI fold All of

the FMN-dependent proteins in this clan serve as

elec-tron transfer proteins or act as two-elecelec-tron reductases

for free flavin (FMN reductase, EC 1.5.1.29) or other electron acceptors (e.g azobenzene reductase,

EC 1.7.1.6) In addition to these two most abundant structural clans, FMN-dependent proteins are found in

12 rare folds Some of these folds are unique struc-tures, and are found in only one or a few enzymes, such as bacterial luciferase (Bac_luciferase), nitroreduc-tase (Nitroreducnitroreduc-tase fold), phosphopantothenate-cyste-ine ligase (clan NADP_Rossmann⁄ family DFP), and chorismate synthase (chorismate_syn) The latter two examples are very interesting, because these two enzymes do not catalyze net redox reactions and are not classical oxidoreductases, like most flavin-depen-dent enzymes (Fig 2) This observation suggests that FMN-dependent enzymes used for ‘aberrant’ activities have evolved independently from the canonical FMN-dependent oxidoreductases, or, in other words, the folds necessary to carry out the enzymatic reaction were not ‘borrowed’ from the oxidoreductases, but instead novel topologies have arisen during the evolution of these enzymes As will be discussed below, this tendency for unusual reactions to call for unusual folds is also found in FAD-dependent enzymes The topologies found for FAD binding are dominated by the Rossmann fold or variations thereof,

FAD

0

10

20

30

40

50

60

70

acyl-CoA_dh FAD_DHS

Flavoprotein FMN-binding

FMN

16

12

6

4 4 4

0

2

4

6

8

10

12

14

16

TIM_barrel Flavoprotein

FMN-binding Bac_luciferase Nitroreductase

A

B

Fig 3 Bar plot of the distribution of structural clans (according to

the PFAM classification) in FMN-dependent (A) and FAD-dependent

(B) flavoproteins.

Fig 4 Graphical representation of the two most common struc-tural clans for FMN-dependent (A, B) and FAD-dependent (C, D) proteins The examples show the structures of flavodoxin from (A) Desulfovibrio vulgaris (PDB entry 1fx1), (B) bold yellow enzyme from Sa cerevisiae (PDB entry 1oyc), (C) glutathione disulfide reductase (PDB entry 3grs), and (D) UDP-N-acetylmuramate dehy-drogenase (PDB entry 1mbt), representing the clans TIM_barrel, Flavoprotein, NADP_Rossmann, and FAD_PCMH, respectively The structure representations were generated with PYMOL

contained in the clan NADP_Rossmann (Fig 4C) [56].

This structure clan comprises a large number of

families (148), with nine families reported to serve for

FAD binding Almost half of the FAD-dependent

pro-teins exhibit a fold in this clan (Fig 3, bottom panel)

Second to the clan NADP_Rossmann is the clan

FAD_PCMH (two families; for a graphical example,

see Fig 4D), followed by the clan FAD_Lum_binding

(five families) and the clan Acyl-CoA_dh (four

fami-lies) Together, the structures found in these four clans

account for 75% of all FAD-dependent proteins The

clans that are rare appear to occur predominantly in

proteins with special biological functions, such as

light-dependent DNA repair (deoxyribodipyrimidine

photolyase,EC 4.1.99.3), oxidoreductase activity in the

endoplasmic reticulum (ERO1), or electron transfer

from acyl-CoA dehydrogenases to the electron

trans-port chain (clan 4Fe–4S) As discussed above for

FMN-dependent proteins, this observation suggests

that employment of FAD-dependent enzymes for novel

or unusual functions requires the adaptation of already

existing topologies and, in some cases, new structural

designs to fulfill the desired role

The majority of covalently bound flavins are present

as FAD rather than FMN (Table S2) Interestingly,

covalent attachment of FAD occurs only in the

two most abundant clans, NADP_Rossmann and

FAD_PCMH, and is almost equally distributed between

these two clans (Table S2) Several families in the clan

NADP_Rossmann are associated with covalent FAD

linkage (DAO, GMC_oxred_N, FAD_binding_2,

Amino_oxidase, and Trp_halogenase) This is in

con-trast to the clan FAD_PCMH, where covalent linkage is

found in the family FAD_binding_4 but not in the

fam-ily FAD_binding_5, which comprises FAD-containing

and molybdopterin-containing enzymes, such as

xan-thine oxidase (EC 1.1.3.22) and

quinoline-2-oxidoreduc-tase (EC 1.3.99.17), to mention only two representatives

of this family (Table S1) Covalent linkage is highly

prevalent in the family FAD_binding_4: 11 of the 14

structures reported for this family show monocovalent

or bicovalent flavin attachment, with

UDP-N-acetyl-muramate dehydrogenase (EC 1.1.1.158), D-lactate

dehydrogenase (EC 1.1.1.28) and

alkyldihydroxyace-tone phosphate synthase (EC 2.5.1.26) being the only

exceptions (Table S2)

Impact of structural genomics

consortia

Several structural genomics projects on prokaryotic

and eukaryotic species have been initiated, in order to

define the structures of expressed proteins in the target

organism A total of 173 (86 for FMN-utilizing pro-teins and 87 for FAD-utilizing propro-teins) entries have been deposited by structural genomics consortia since

1999, amounting to 10% of the total entries ( 1800 entries; 640 for FMN-utilizing proteins and 1160 for FAD-utilizing proteins) Analysis of the structural classification for FMN-dependent proteins reveals a strong bias towards the clan Nitroreductases, with

a total of 27 entries ( 31%) As this clan has only a moderate frequency among FMN-dependent proteins (Fig 3, top panel), this overrepresentation suggests that this type of structure is favored by the methodolo-gies currently used in structural genomics pipelines The aim of the consortia to elucidate the structures of

as many different proteins as possible also leads to a serious lack of biochemical information, which renders some of the PDB entries difficult to interpret in terms

of the biological function of the flavoprotein On the other hand, several structures of new flavoproteins with unknown roles have been contributed by struc-tural genomics initiatives For example, a zinc-depen-dent protease from Bacteroides thetaiotaomicron (clan Glutaminase_I, family DJ-1⁄ PfpI, PDB entry3cne) and protein structures with a fold similar to the C-ter-minal domain of pyruvate kinase in the archaeons Archaeoglobus fulgidus and Methanobacterium thermo-autotrophicum were recently deposited in the PDB (clan PK_C, PDB entries1vp8 and 1t57) However, the role of the FMN cofactor in these two proteins is unclear In the putative protease, the flavin isoalloxa-zine ring is sandwiched by two tryptophans at the interface of the dimeric protein, with the edge of the pyrimidine ring moiety at distance of 15 A˚ from the presumably catalytic mononuclear zinc center Hence, the flavin does not appear to play a role in catalysis, but may instead be involved in dimerization of the protein or act as a gate for potential substrates to enter the active site On the other hand, the flavin in the pyruvate kinase fold in archaeons is located in a central cavity of the protein, and engages in hydrogen bond interactions with several amino acid side chains

In this case, it seems plausible that the flavin plays a catalytic role, albeit in a type of fold that has not previously been implicated in flavoenzyme catalysis Furthermore, an FMN-dependent oxidoreductase from Thermotoga maritima was the first structure of a flavin-dependent tRNA dihydrouridine synthase (clan TIM_barrel, family Dus; PDB entry1vhn),

an enzyme that has recently been characterized biochemically [60]

In the case of FAD, the entries provided by struc-tural genomics consortia reflect the predominance of the clan NADP_Rossmann, with 44 of 87 entries

belonging to this clan Interestingly, several new

struc-tural families for FAD-dependent proteins were

defined in the course of structural genomics efforts,

such as the bluf domain of blue light sensors in

cyano-bacteria (1x0p), the glucose-inhibited division

pro-tein A (GidA) domain in the clan NADP_Rossmann,

the HI0933-like proteins (first discovered in target 0933

from Haemophilus influenzae, PDB entry2gqf), and a

siderophore-interacting protein (family

FAD_bind-ing_9 in the clan FAD_Lum_binding) In addition, a

novel covalent attachment between a side chain

car-boxylate group of an aspartate and the 8a-position of

the isoalloxazine system was discovered in an

FAD-dependent halogenase involved in chloramphenicol

biosynthesis in Streptomyces venezuelae [47] As noted

before, this structural information provides interesting

leads for biochemists to follow up and subject these

proteins to thorough biochemical characterization in

order to reveal their cellular role

Flavogenomics – occurrence and

distribution of flavoproteins in

prokaryotes and eukaryotes

Despite the availability of genomic sequence

informa-tion, it proved difficult to obtain reliable information

on the occurrence of flavoproteins encoded in the

genomes of various organisms This is mostly because

of the lack of information on whether a flavin (FMN

and⁄ or FAD) cofactor is present and the precise

biochemical reaction catalyzed by the enzyme On the

other hand, it is doubtful that all, or even most, of the

proteins predicted by genomics will ever be subjected

to a detailed characterization that would enable

accu-rate functional assignment of a putative flavoenzyme

For most of the species analyzed, we used the

annota-tions provided by the responsible sequencing facility,

and included only those entries that gave a clear

indication of flavin dependence (see Methods) This

approach probably leads to an underestimation of the

number of flavoproteins, as many ‘hypothetical’ or

‘putative’ proteins may be flavin-dependent but are not

annotated as such An interesting alternative to use of

the existing annotations is the analysis of predicted

protein families as provided by the Broad Institute

for Neurospora crassa (http://www.broadinstitute.org/

annotation/genome/neurospora/Pfam.html) and on the

tuberculosis research platform for Mycobacterium

tuberculosis and Streptomyces coelicolor (http://www

tbdb.org/) Therefore, we have also used our set of

structural families (Table S3) to search for proteins

predicted in the above-mentioned species In the case

of M tuberculosis, a parallel analysis of the available

genome annotation was conducted The ‘structural family approach’ has generated a significantly higher number of predicted flavoproteins (141 versus 113), as many hypothetical proteins are found in protein families that are typical or even specific for flavopro-teins (e.g FAD_binding_4 or NPD) and hence were included as predicted flavin-dependent proteins The disadvantage of this more ‘inclusive’ analysis is that some of the protein families, such as PAS_3, are not specific for flavin and may utilize other cofactors (e.g heme) In any case, the task of eliminating the false positives and false negatives inherent in both approaches can only be performed by biochemical characterization of predicted and suspected flavopro-teins To this end, structural genomics may also play

an important role; however, flavoenzymes that do not hold on tightly to the flavin cofactor (e.g chorismate synthase) or use it only transiently during catalysis (e.g hydroxypropylphosphonic acid epoxidase) may elude identification as flavin-dependent proteins Although it is presently not possible to determine the exact number of flavoproteins, our analysis has revealed striking differences in the utilization of flavin-dependent proteins in various prokaryotic and eukaryotic species, which are reflected both by the total number and the percentage of genes encoding flavoproteins (Fig 5) Several species appear to have a minimum number of flavin-dependent proteins that are required to maintain basic metabolic functions, such as succinate dehydrogenase which is necessary for pri-mary energy metabolism, and chorismate synthase and acetolactate synthase, which are necessary for amino acid biosynthesis Examples of species with a minimal set of enzymes are Pyrococcus abyssi, T maritima, and Saccharomyces cerevisiae (with 12, 12 and 48 entries, respectively) On the other hand, organisms such as

M tuberculosis, Neurospora crassa, S coelicolor and Arabidopsis thaliana contain a relatively large number

of genes encoding flavin-dependent proteins In these cases, flavoenzymes are apparently involved in a species-specific lifestyle that requires a much larger set

of flavoenzymes than are needed by the ‘flavin mini-malists’ mentioned before Closer inspection of the set

of flavoenzymes in these organisms reveals a multitude

of one or several types of flavin-dependent proteins In order to estimate this redundancy of a ‘flavogenome’,

we have defined the quotient of the number of distinct flavin-dependent proteins (i.e with different EC numbers) and the total number of flavin-dependent proteins as a ‘redundancy’ index (RI) (RI = 1 indi-cates a nonredundant flavogenome, whereas RI < 1 indicates increasing redundancy; Fig 5C) In the case

of M tuberculosis,  34 genes encoding acyl-CoA

dehydrogenases and  10–15 genes encoding flavin-containing monooxygenases and oxidoreductases give rise to high redundancy (RI = 0.55; Fig 5C) The occurrence of this many acyl-CoA dehydrogenases is apparently related to the extensive and complex utiliza-tion of lipids from host cells by this pathogenic bacte-rium [61] A large number of genes encoding acyl-CoA dehydrogenases is also found in S coelicolor, and is only exceeded by putative flavin-dependent oxidore-ductases, with 57 predicted genes Again, the abundance of these flavoenzymes can be rationalized

on the basis of the lifestyle: S coelicolor is a rather immobile soil bacterium that can adapt to various car-bon and nitrogen sources and produces a large number

of biologically active compounds, such as antibiotics

In other words, the organism depends on metabolic power and versatility that are certainly conferred to some degree by flavin-dependent enzymes In contrast

to M tuberculosis and S coelicolor, N crassa has apparently pursued a different metabolic strategy by using a broader array of flavoenzymes rather than a highly similar set, as indicated by the rather high RI (0.74 versus 0.5 and 0.55 for S coelicolor and

M tuberculosis, respectively) As a result, N crassa contains more than 100 different flavoproteins, more than any other species analyzed in our study The large number of flavoenzymes in this filamentous fungus may be attributable to diverse biosynthetic routes leading to secondary metabolites, as well as the sapro-trophic lifestyle, which requires the generation and secretion of oxidases and dehydrogenases to access organic matter in the environment In this context, it is noteworthy that the protein family FAD_binding_4 constitutes the largest group among the predicted puta-tive flavoenzymes in this species Members of this fam-ily are typically oxidases that are capable of performing a wide range of substrate (e.g sugars and alcohols) oxidation reactions [48]

The flavogenome of the model plant A thaliana is the most prolific among the analyzed genomes This is mostly because of the occurrence of two large groups

of flavoproteins, monooxygenases and oxidases of the (S)-tetrahydroprotoberberine oxidase⁄ berberine bridge

0

50

100

150

200

250

P abyssi B subtilis

C trachomatis De radiodurans

M tuberculosis Ps aeruginosa

S coelicolor T maritima

To gondii N crassa

Sa cerevisiae A thaliana

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

P abyssi B subtilis

S coelicolor T maritima V fischeri

To gondii N crassa

P abyssi B subtilis

M tuberculosis Ps aeruginosa

To gondii N crassa

1

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0

A

B

C

Fig 5 Occurrence and distribution of flavoproteins in 22 selected genomes (A) The number of genes encoding flavin-dependent pro-teins in the genomes of My genitalium, Ar fulgidus, Me janaschii,

P abyssi, Pl falciparum, To gondii, Sa cerevisiae, N crassa,

A thaliana, D melanogaster and Homo sapiens (B) The numbers

of predicted flavoproteins as percentages of the total proteins for the species in (A) (C) The RIs of flavoproteins in these genomes Yellow bars indicate genomes with low redundancy, and brown bars indicate genomes with high redundancy.

enzyme family, with 31 and 26 members, respectively.

As previously discussed for microbial genomes, the

large number of enzymes in these two flavoprotein

families is a reflection of the diversity of metabolic

processes employed to synthesize a vast array of

bioac-tive compounds In the case of plants, natural products

such as alkaloids and terpenes are among the

com-pounds synthesized for signaling and defense purposes

Several members of the berberine bridge enzyme family

are implicated in plant metabolism, such as

(S)-tetra-hydroprotoberberine oxidase, nectarin V [62], and

pol-len allergen proteins [63] Therefore, it can be expected

that most of the flavoproteins occurring in these two

groups will catalyze distinct reactions on various

dif-ferent substrates

The RI seems to be a useful tool with which to

iden-tify organisms that have a ‘flavin-dependent’ lifestyle

because of their high demand for chemically complex

biomolecules, and which are thus potentially

vulnera-ble to inhibitors of riboflavin biosynthesis and⁄ or

uptake [64–66] Although it is apparent that major

spe-cies-specific differences exist, the currently estimated

RIs are probably too low for several species, owing to

the lack of biochemical knowledge of the enzymes in

the most common flavoprotein families Hence, future

efforts to define the flavoprotein arsenal of an

organ-ism have to focus on three aspects: to capture all true

flavin-dependent proteins, to eliminate false positives,

and to characterize the flavoproteins biochemically in

order to classify them accurately As a significant first

step, it would be useful to conduct an HMMER

analy-sis [67] of the existing genomes to provide a list of

potential flavoproteins, to enable scientists to target

specifically these putative genes for biochemical and

structural studies

Methods

Flavoproteins from different species were identified by

screening pertinent databases Microbial genomes were

analyzed by screening the databases provided by the J Craig

Venter Institute (Ar fulgidus DSM4304, Bacillus

subtil-is168, Chlamydia trachomatis serovar D, Deinococcus

radio-duransR1, Escherichia coli K-12, Helicobacter pylori 26695,

Methanocaldococcus jannaschii, M tuberculosisCDC1551,

Mycoplasma genitaliumG-37, Pseudomonas aeruginosa PAO,

P abyssi, Staphylococcus aureus MW2, T maritima, and

Vibrio fischeriES114) Putative flavoproteins in N crassa

were retrieved by a web-based analysis of the known

flavin-dependent protein families listed in Table S1 on

http://www.broadinstitute.org/annotation/genome/neurospora/

MultiHome.html Flavoproteins in the yeast Sa cerevisiae

were identified with the annotations available on the yeast

genome website at http://www.yeastgenome.org A similar approach was used for M tuberculosis and S

coelicol-orA3(2) (http://www.tbdb.org/) Information on flavopro-teins in the human parasites Plasmodium falciparum and Toxoplasma gondii were retrieved by inspection of http:// plasmodb.org/plasmo/ and http://toxodb.org/toxo, respec-tively Flavoproteins from A thaliana were retrieved by a keyword and protein name search (flavin, FMN, FAD, diox-ygenase, monooxdiox-ygenase, hydroxylase, and the individual names of all flavoproteins listed in Table S1), with the ARa-bidopsis Gene EXpression Database (AREX) (http:// www.arexdb.org/index.jsp) Analysis of flavoproteins in Dro-sophila melanogaster was based on a search in http://fly-base.org/ and http://www.brenda-enzymes.org Human flavoproteins were identified by a text search with the enzyme names from Table S1 in the Online-Mendelian Inheritance in Man (OMIM) database (http://www.ncbi nlm.nih.gov/omim)

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Supporting information

The following supplementary material is available: Table S1 List of flavin-dependent proteins (FMN, FAD, riboflavin and derivatives)

Table S2 Covalent attachment of FMN and FAD Table S3 Protein families (PFAM) used for structural classification of flavin-dependent proteins

This supplementary material can be found in the online version of this article

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