Gene annotation: Laccases and multicopper oxidases

P.J. Hoegger1, S. Kilaru1, P.E. Courty2, F. Martin2, U. Kües1

1Georg-August-University Göttingen, Germany2INRA Nancy, France

4th Workshop of the Laccaria Genome Consortium

Georg-August-Universität, Göttingen - November 16-17, 2006

Multi-copper oxidase family

main members: - laccases

- fungi

- plants

- insects

- bacteria

- ascorbate oxidases- plants

- ferroxidases- fungi

- ceruloplasmins- mammals

Laccase function and application

Biological functions: - lignin degradation

- lignification

- pigment synthesis

- fruiting body formation

- melanin polymerization

- detoxification

- cuticle sclerotization

- soil litter polymer degrad.

Applications: - bleaching processes

- bioremediation

- composite wood production

Trametes versicolorlaccase Lcc1 Piontek et al. 2002, JBC 277, 37663-37669

1 type-1 Cu

1 type-2 Cu2 type-3 Cu

Fungal laccase structure

L1 L2 L4L3

Signal sequence

signature sequences, including copper ligands

H H H H H H H HCH H

Substrate binding loops

I II III IV

Scaffold 39

lcc11 lcc1262 kb222 kb 25 kb

lcc273 kb 175 kb

lcc3112 kb 3166 kb

lcc4194 kb 904 kb

lcc5116 kb 312 kb

lcc6429 kb

lcc7 lcc8

0.53 kb0.53 kb569 kb

lcc9143 kb 110 kb

lcc10647 kb 65 kb

Scaffold 69 Scaffold 2

Scaffold 13 Scaffold 47

Scaffold 67 Scaffold 27

Scaffold 14

MCOs in the genome of L. bicolor

Species MCOmembers

C. cinerea 17

C. neoformans 6

U. maydis 6

P. chrysosporium 5

F. graminearum 13

M. grisea 11

N. crassa 10

A. nidulans 7*

C. globosum 7

C. immitis 2

R. oryzae 5

yeasts 3-5

S. pombe 1

group nr. sequences

basidiomycetes 146

ascomycetes 108

zygomycetes 5

plants 62

animals 12

bacteria 29

total 362

Multi-copper oxidasephylogenetic tree

basidiomycetelaccases

ascomycetelaccasesinsect laccases

Cel NP 501502

fungal pigment MCOs

fungal ferroxidases

Mgr Mco7 Sce AAB64948

Cgl XP 448078 Kla XP 452271

ascorbate oxidases

"ferroxidases / laccases"

Pch AAO42609 MCO1 Pch AAS21669 MCO4 Pch AAS21659 MCO2

Pch AAS21662 MCO3 Mgr Mco1

Fgr Mco1 Uma Mco1 Ego NP 984335

Uma Mco3 Cne Mco5

Cne A36962 Cne Mco6 Cim Mco2

Fgr Mco10

0.1bacterial MCOs

plant LMCOs

laccases

sen

su

str

icto

basidiomycetelaccases

ascomycetelaccasesinsect laccases

Cel NP 501502

fungal pigment MCOs

fungal ferroxidases

Mgr Mco7 Sce AAB64948

Cgl XP 448078 Kla XP 452271

ascorbate oxidases

"ferroxidases / laccases"

Pch AAO42609 MCO1 Pch AAS21669 MCO4 Pch AAS21659 MCO2

Pch AAS21662 MCO3 Mgr Mco1

Fgr Mco1 Uma Mco1 Ego NP 984335

Uma Mco3 Cne Mco5

Cne A36962 Cne Mco6 Cim Mco2

Fgr Mco10

0.1bacterial MCOs

plant LMCOs

laccases

sen

su

str

icto L. bicolor MCOs

in phylogenetic tree

LbiLcc1 LbiLcc2

LbiLcc3 LbiLcc4

LbiLcc5 LbiLcc6

LbiLcc7 LbiLcc8

LbiLcc9

LbiLcc11

LbiLcc10

Tve B35883 ligninolytic phenol . Thi Q02497 laccase .

AH2 AAW28933 lacA . I62 AAB63444 Pox2 .

Thi AAL89554 072-1 . Tpu AAM18407 Lap2 . Tve AAL07440 Lac1 .

Tvi Q99044 LCC1 . Tsp-420 ABB21020 lacE .

Ftr CAC13040 Lcc1 . PM1 CAA78144 laccase . C30 AAF06967 LAC1 . Cga ABD93940 lac1 .

Pci AAG13724 Lac1 . Pco BAB69775 Lcc1 . Pci AAC39469 Lcc3-1 .

Pcl AAG09229 Lcc3-1 . Tts BAA28668 bilirubin oxidase .

I62 AAB63445 Pox3 . Tsp-AH AAW28934 lacC .

Fve AAR82931 laccase . Glu AAR82934 laccase .

Pci AAD49218 Lcc3-2 . Psa AAR20864 laccase .

I62 AAQ12267 Pox1 . Tvi Q99046 LCC2 . Tve AAW29420 lcc1 .

Tve Q12717 LCC5 . Tvi Q99056 LCC5 .

Tpu AAM18408 Lap1A . Tsp-AH AAW31597 lacB .

C30 AAM66349 Lac2 . 420 AAW28936 lacA .

Pcl AAG09230 Lcc3-2 . Tve Q12719 LCC4 .

Tvi Q99055 LCC4 . 420 AAW28939 lacD .

C30 AAR00925 Lac3 . Csu AAC97074 Lcs1 .

Pra CAA36379 Lac . Ptr CAK54346 lac1 .

Led BAB83131 LeLcc1 . Pru AAW28932 lacA .

Ssp-FERM BAE96003 Lac2 . Ssp-FERM BAE79811 Lac1 .

420 AAW28937 lacB . Tvi Q99049 LCC3 .

Cga AAF70119 Lcc1 . Rmi AAO38869 Lcc .

Rmi CAE81289 lcc1 . Pos CAA06292 PoxA1b .

Per ABB30169 pel4 . Psc CAD45380 Lac4 . Ppm AAX40733 lac2 .

Pos Q12739 POX2 . Psp CAH05069 lac1 . Psc CAD45377 Lac1 .

Pos Q12729 POX1 . Per AAV85769 pel3 .

Psc CAD45378 Lac2 . Psc CAD45381 Lac5 .

Lbi Lcc7 . Lbi Lcc8 .

Lbi Lcc6 . Led AAF13037 Lac1 .

Led BAC06819 LeLcc3 . Cci BK004118 Lcc8 .

Sco BAA31217 laccase . Vvo AAR03582 lac3 .

Cci BK004120 Lcc10 . Cci AAR01246 Lcc5 .

Cci BK004119 Lcc9 . Cci FA2222 lcc15 .

Cci BK004114 Lcc4 . Cci AY464531 Lcc1 .

Cci BK004116 Lcc6 . Cci AAR01248 Lcc7 .

Cci BK004121 Lcc11 . Lbi Lcc4 .

Lbi Lcc1 . Lbi Lcc5 .

Lbi Lcc2 . Lbi Lcc3 .

Lbi Lcc9 . Cci BK004122 Lcc12 . Cci BK004123 Lcc13 .

Cco CAB69046 Clac2 . Cci BK004112 Lcc2 .

Cci AAD30966 Lcc3 . Cci BK004124 Lcc14 .

Fve BAE80732 lcc2 . Vvo AAR03583 lac5 . Vvo AAR03584 lac4 .

Vvo AAR03581 lac2 . Vvo AAO72981 Lac1 .

Vvo AAR03585 lac6 . Hvi AAZ06799 lcc1 .

Abi Q12541 LCC1 . Abi Q12542 LCC2 .

Tesp-NM BAE53769 lcc1-2 . Pos CAC69853 Poxa3 . Psc CAD45379 Lac3 . Ppm AAX40732 lac6 .

Tesp-NM BAE53770 lcc2-5 . Cci BK004126 Lcc16 .

Cci BK004127 Lcc17 . Rso S6812 laccase 4 .

Rso Q02075 LCC2 . Rso P56193 LCC1 .

Rso Q02079 LCC3 .100

100

100

91100

100

100

67

77

84

92100

73

92

92

5092

100

100

72

100

98

99

100

95

100

99

100

3979

5294

6188

100

100

99

37

97

78

98

100

100

90

75

88

97

100

92100

468999

4499

99

80100

99

100

89

65

96

85

67

98

85

60

62

90

59

29

33

73

27

91

96

29

27

59

30

66

59

32

13

27

32

67

41

50

40

28

57

5

3

7

16

3

5

547

14

18

9

19

6

7

0.05

wood decaying species(Aphyllophorales, Agaricales)

wood and litter decomposers(Agaricales)

Intron position conservation in basidiomycete laccase genes

Tree based on intron positions

Scaffold 39

lcc11 lcc1262 kb222 kb 25 kb

lcc273 kb 175 kb

lcc3112 kb 3166 kb

lcc4194 kb 904 kb

lcc5116 kb 312 kb

lcc6429 kb

lcc7 lcc8

0.53 kb0.53 kb569 kb

lcc9143 kb 110 kb

lcc10647 kb 65 kb

Scaffold 69 Scaffold 2

Scaffold 13 Scaffold 47

Scaffold 67 Scaffold 27

Scaffold 14

L. bicolor MCO gene family

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1_2

1_3

1_4

1_5

1_6

1_7

1_8

1_9

2_3

2_4

2_5

2_6

2_7

2_8

2_9

3_4

3_5

3_6

3_7

3_8

3_9

4_5

4_6

4_7

4_8

4_9

5_6

5_7

5_8

5_9

6_7

6_8

6_9

7_8

7_9

8_9

pairs of genes (L. bicolor laccases)

no

. of

ob

se

rve

d c

ha

ng

es

/ n

o. o

f p

ote

nti

al c

ha

ng

es

synonymous rates

non-synonymous rates

Syn. / non-syn. substitution rates

basidiomycetelaccases

ascomycetelaccasesinsect laccases

Cel NP 501502

fungal pigment MCOs

fungal ferroxidases

Mgr Mco7 Sce AAB64948

Cgl XP 448078 Kla XP 452271

ascorbate oxidases

"ferroxidases / laccases"

Pch AAO42609 MCO1 Pch AAS21669 MCO4 Pch AAS21659 MCO2

Pch AAS21662 MCO3 Mgr Mco1

Fgr Mco1 Uma Mco1 Ego NP 984335

Uma Mco3 Cne Mco5

Cne A36962 Cne Mco6 Cim Mco2

Fgr Mco10

0.1bacterial MCOs

plant LMCOs

laccases

sen

su

str

icto L. bicolor MCOs

in phylogenetic tree

LbiLcc1 LbiLcc2

LbiLcc3 LbiLcc4

LbiLcc5 LbiLcc6

LbiLcc7 LbiLcc8

LbiLcc9

LbiLcc11

LbiLcc10

Iron uptake in fungi

Kosman 2003, Mol Microbiol, 47:1185-1197

ftr1a

ftr1b lcc11

lcc10

lcc10 and lcc11 loci

nucleotide id. 89% 86%

aa id. / sim. 91% / 95% 84% / 88%

331 bp 115 bp179 bp

205 bp

iron permease ferroxidase

observed syn. sub.

potential syn. sub.

ratio

observed non-syn. sub.

potential non-syn. sub. ratio

ftr1a / ftr1b

52 280 0.19 38 890 0.04

lcc10 / lcc11

81 410 0.20 53 1318 0.04

Syn. / non-syn. substitution rates of lcc10/lcc11 and ftr1a/ftr1b

Intron position conservation in basidiomycete ferroxidase genes

MCO arsenal inbasidiomycete species

Cci Lbi Pch Cne Uma

Total MCOs 17 11 5 6 6

Laccases s. str. 17 9

Ferroxidases 2 1 4 1

Pigment MCOs 2

Ascorbate oxidases 1

Ferroxidases / laccases 4 2 2

MCO arsenal inbasidiomycete species

Cci Lbi Pch Cne Uma

Total MCOs 17 11 5 6 6

Laccases s. str. 17 9

Ferroxidases 2 1 4 1

Pigment MCOs 2

Ascorbate oxidases 1

Ferroxidases / laccases 4 2 2

High affinity iron uptake pathway

ftr1 homologue(s)

ftr1 clustered with MCOs - 2 1 2 1

sid1 / sidA homologues

MCO expression profile

MCO gene L 0.5 % L 2 % W 0.1 % W 0.4 % Fb M

lcc1 1.60 ± 0.23 4.97 ± 0.26 2.59 ± 0.43 2.68 ± 0.03 1.60 ± 0.20 5.68 ± 0.88

lcc2 1.97 ± 0.26 1.01 ± 0.16 1.08 ± 0.14 3.07 ± 0.56 1.50 ± 0.17 7.56 ± 0.81

lcc3 1 1 1 1 1 1

lcc4 0.94 ± 0.13 0.64 ± 0.25 0.80 ± 0.18 0.91 ± 0.05 0.65 ± 0.15 0.33 ± 0.08

lcc5 0.87 ± 0.13 0.86 ± 0.13 0.44 ± 0.09 2.38 ± 0.34 1.63 ± 0.22 0.59 ± 0.24

lcc6 1.09 ± 0.31 0.98 ± 0.33 1.34 ± 0.13 1.13 ± 0.35 1.21 ± 0.38 0.46 ± 0.03

lcc7 1.29 ± 0.20 0.67 ± 0.36 1.19 ± 0.19 1.17 ± 0.34 14.49 ± 1.18 0.69 ± 0.09

lcc8 1.04 ± 0.28 0.69 ± 0.11 0.32 ± 0.10 0.95 ± 0.19 0.76 ± 0.23 0.56 ± 0.04

lcc9 1.30 ± 0.44 0.86 ± 0.22 1.02 ± 0.24 0.79 ± 0.12 1.72 ± 0.24 0.21 ± 0.12

lcc10 3.83 ± 0.16 1.79 ± 0.51 3.10 ± 0.45 1.27 ± 0.23 1.26 ± 0.26 0.54 ± 0.09

lcc11 1.27 ± 0.50 0.32 ± 0.19 0.36 ± 0.08 3.45 ± 0.78 0.22 ± 0.03 1.49 ± 0.28

AcknowledgementsMolecular Wood Biotechnology, Institute of Forest Botany, Georg-August-

University Göttingen, Germany

Ursula Kües

Patrik Hoegger

Sreedhar Kilaru

UMR 1136 Interactions Arbres Micro-organismes, Centre INRA de Nancy,

Champenoux, France

Francis Martin

Marc Buée

Pierre-Emmanuel Courty

MCO arsenal in fungal species

basidiomycetes zfilamentous ascomycetes ascomycetous yeasts

Cluster

Cci Cne Pch Uma Ani Ncr Fgr Mgr Cgo Cim Ego Sce Cal Cgl Cgu Clu Ctr Kla Dha Yli Spo Ror

Total MCOs 17 6 5 6 7 10 13 11 7 2 3 3 5 3 3 3 3 3 3 3 1 5 Basidiomycete laccases 17 Ascomycete laccases 2 8 5 4 4 1 Ferroxidases / laccases 2 4 2 1 1 Ferroxidases 4 1 1 2 3 3 2 3 2 5 2 2 2 3 2 2 3 1 1 Ascorbate oxidases 1 2 1 1 1 4 Conidial pigment MCOs 2 3 3 2 1 1 1 Not in any cluster 1 1 1 1 Genes of high affinity iron uptake pathways ftr1 homologue(s) ftr1 clustered with MCOs - 2 1 1 - 1 2 1 1 - - - - - - - - - - 1 1 1 sid1 / sidA homologues

ftr1a

ftr1b lcc11

lcc10

lcc10 and lcc11 loci

nucleotide id. 89% 86%

aa id. / sim. 91% / 95% 84% / 88%

frame-shift? A>G

A>GT/C>GG>A

A/C>T

331 bp 115 bp179 bp

205 bp