Natalie D. Fedorova

1
Functional diversification and evolution of
biotin-dependent enzymes
Natalie D. Fedorova
2
Introduction

• protein classification databases are essential
  for functional annotation of proteins in the
  course of large-scale genome sequencing projects
• one of the factors that significantly complicate
  the automatic functional annotation is the
  existence of multi-domain proteins
• because of the multi-domain organization
  straightforward sequence comparisons do not
  always allow for accurate functional predictions
• non-similarity based approaches, unified under
  the term genomic context can be used to improve
  functional assignments of complex proteins and
  multi-component systems, such as biotindependent
  enzymes

3
Outline

• range of the reactions catalyzed by BDE
• domains/subunits reactions catalyzed by
  subunits
• operon organization of the corresponding genes
• biotin-dependent enzyme from Giardia intestinalis
  
• 3 classes of BDE domain/subunit organization
• phylogenetic analysis of BDE
• genomic context of the G. intestinalis BDE
• conclusions

4
Reactions catalyzed by biotin-dependent enzymes
? several key metabolic reactions in
gluconeogenesis and fatty acid biosynthesis,
hydrolysis of urea to ammonia and carbon dioxide,
the leucine catabolism pathway, microbial
degradation of benzoyl-CoA and other aromatic
compounds, and in energy transformation ?
carboxylases, decarboxylases and
transcarboxylases ? recognize a variety of
substrates acetate, malonate, propionate,
methylmalonate, pyruvate and oxaloacetate ?
share a similar catalytic mechanism involving 2
partial reactions carboxylation of biotin
moiety covalently attached to BCCP carboxyl
transfer from biotin to the acceptor molecule
5
Outline

• range of the reactions catalyzed by BDE
• domains/subunits reactions catalyzed by
  subunits
• operon organization of the corresponding genes
• biotin-dependent enzyme from Giardia intestinalis
  
• 3 classes of BDE domain/subunit organization
• phylogenetic analysis of BDE
• genomic context of the G. intestinalis BDE
• conclusions

6
Domains/subunits of biotin-dependent
enzymes BCCP, biotin carboxylase, OadBG, and
carboxyltransferase (ACCT and PycB) ? ACCT
acetyl-CoA carboxyltransferase consists of two
subdomains A and D ? PycB propionyl-CoA
carboxyltransferase
BCCP
Pyruvate/oxaloacetate transcarboxylase
Acetyl-CoA carboxylase alpha
Acetyl-CoA carboxylase beta
Membrane-bound subunit OadB
OadG
Biotin carboxylase
7
Reactions catalyzed by subunits of
biotin-dependent enzymes carboxyl transfer from
biotin to the acceptor molecule
Biotin carboxylase (present in BDE involved in
CO2 assimilation) HCO3- ATP BCCP ??
-COO-BCCP ADP Pi AccAAccD-type
carboxyltransferase -COO-BCCP acetyl-CoA ??
BCCP malonyl-CoA -COO-BCCP propionyl-CoA ??
BCCP methylmalonyl-CoA -COO-BCCP crotonyl-CoA
?? BCCP glutaconyl-CoA -COO-BCCP
methylcrotonyl-CoA ?? BCCP methylglutaconyl-CoA
PycB/OadA-type carboxyltransferase -COO-BCCP
pyruvate ?? BCCP oxaloacetate OadB subunit of
membrane-bound decarboxylases -COO-BCCP ? BCCP
HCO3- DmNa
8
Outline

• biotin-dependent enzyme from Giardia intestinalis
• range of the reactions catalyzed by BDE
• domains/subunits reactions catalyzed by
  subunits
• operon organization of the corresponding genes
• 3 classes of BDE domain/subunit organization
• phylogenetic analysis
• genomic context of the Giardia BDE
• conclusions

9
E. coli acetyl-CoA carboxylase 4 separate genes
In most cases BCCP is found fused to the biotin
carboxylase or carboxyl transferase domains.
Although in some prokaryotes, BCCP is encoded by
a separate gene accB that may or may not be a
part of an operon encoding other subunits.
AccC - Biotin carboxylase
BCCP
AccA - Acetyl-CoA carboxylase alpha
AccD - Acetyl-CoA carboxylase beta
Biotin carboxylase HCO3- ATP BCCP ??
-COO-BCCP ADP Pi
AccAAccD-type carboxyltransferase
-COO-BCCP acetyl-CoA ?? BCCP malonyl-CoA
-COO-BCCP propionyl-CoA ?? BCCP
methylmalonyl-CoA
10
Biotin-dependent enzyme from Giardia
intestinalis ? annotated as pyruvate
carboxylase ? N-terminal portion is similar to
acetyl-CoA and propionyl-CoA carboxylases,
C-terminal portion is similar to the pyruvate
carboxylase group (PycB-type) ? BCCP shared
by all biotin-dependant enzymes ?
multi-domain enzyme with a unique domain
organization
BCCP
AccA - Acetyl-CoA carboxylase alpha
AccD - Acetyl-CoA carboxylase beta
Pyruvate/oxaloacetate transcarboxylase
11
Outline

• range of the reactions catalyzed by BDE
• domains/subunits reactions catalyzed by
  subunits
• operon organization of the corresponding genes
• biotin-dependent enzyme from Giardia intestinalis
• 3 classes of BDE domain/subunit organization
• phylogenetic analysis
• genomic context of the Giardia BDE
• conclusions

12
Carboxylases carboxyltransferase domains ?
acetyl-CoA carboxylase (ACCT) 2 subdomains A and
D ? pyruvate carboxylase (PycB) Acetyl-CoA
carboxylases
Acetyl-CoA carboxylase (bacteria)
AccC - Biotin carboxylase
BCCP
AccA - Acetyl-CoA carboxylase alpha
AccD - Acetyl-CoA carboxylase beta
Acetyl-CoA carboxylase (eukaryotes)
Biotin carboxylase
BCCP
AccA - Acetyl-CoA carboxylase alpha
AccD - Acetyl-CoA carboxylase beta
800 aa
domain
Propionyl-CoA carboxylase (eukaryotes)
Beta subunit
Alpha subunit
Biotin carboxylase
BCCP
AccA - Acetyl-CoA carboxylase alpha
AccD - Acetyl-CoA carboxylase beta
13
Pyruvate carboxylases ? pyruvate
carboxyltransferase (PycB) domain
Pyruvate carboxylase (bacteria, eukaryotes)
Biotin carboxylase
Pyruvate/oxaloacetate transcarboxylase
BCCP
Pyruvate carboxylase (archaea)
Biotin carboxylase
Pyruvate/oxaloacetate transcarboxylase
BCCP
Biotin carboxylase HCO3- ATP BCCP ??
-COO-BCCP ADP Pi
PycB-type carboxyltransferase
-COO-BCCP pyruvate ?? BCCP oxaloacetate
14
Methylmalonyl-CoApyruvate transcarboxylase ?
found in Propionibacterium freudenreichii
BCCP
Pyruvate/oxaloacetate transcarboxylase
AccAAccD-type carboxyltransferase
-COO-BCCP propionyl-CoA ?? BCCP
methylmalonyl-CoA
PycB-type carboxyltransferase
-COO-BCCP pyruvate ?? BCCP oxaloacetate
15
Methylmalonyl-CoA decarboxylase
? coupled with the generation of the
transmembrane Na gradient ? principal
mechanism of energy conservation in some
anaerobic bacteria
Methylmalonyl-CoA decarboxylase
BCCP
OadG
Membrane-bound subunit OadB
AccAAccD-type carboxyltransferase
-COO-BCCP propionyl-CoA ?? BCCP
methylmalonyl-CoA
OadB subunit -COO-BCCP ?
BCCP CO2 DmNa
16
Oxaloacetate decarboxylase
? coupled with the generation of the
transmembrane Na gradient ? principal
mechanism of energy conservation in some
anaerobic bacteria
Pyruvate carboxylase
Biotin carboxylase
Pyruvate/oxaloacetate transcarboxylase
BCCP
PycB-type carboxyltransferase
-COO-BCCP pyruvate ?? BCCP oxaloacetate
OadB subunit -COO-BCCP ?
BCCP CO2 DmNa
17
Outline

• biotin-dependent enzyme from Giardia intestinalis
• range of the reactions catalyzed by BDE
• domains/subunits reactions catalyzed by
  subunits
• operon organization of the corresponding genes
• 3 classes of BDE domain/subunit organization
• phylogenetic analysis
• genomic context of the Giardia BDE
• conclusions

18
PycB - BCCP tree
PYCA MYCTU
TR_GIARDIA
55
100
PYCA MLOTI
87
100
PYCA BACSU
PYCA LACLA
PYCA ASPNI
100
100
PYC1 YEAST
PYC2 YEAST
100
PYCA CAEEL
100
100
PYCA AEDES
97
PYCA DROME
PYC HUMAN
100
PYC MOUSE
PYCB PSEAE
69
87
TR_PROFR
OADA TREPA
83
OAD2 VIBCH
100
OADA VIBCH
85
OADA PASMU
98
100
DCOA SALTY
DCOA KLEPN
SPy1174
90
SPy1191
60
PH0834
84
98
aq 1520
99
aq 1614
TM0128
89
AF1252
81
88
PYCB METJA
PYCB METTH
10
19
BCCP tree
100
COAC YEAST
82
II
COA1 HUMAN
COA2 HUMAN
74
DRA0310
PA1400
63
Rv3285
49
OADA TREPA
MMCA SOYBN
83
38
PA2012
II
100
PCCA HUMAN
PCCA MOUSE
96
BCCA MYCTU
70
Rv0973c
100
MMCA HUMAN
MMCA MOUSE
73
VI
BCCP PROFR
74
71
SP1176
RP618
TM0717
75
BCCP PORPU
67
100
I
DR0118
99
89
BCCP ECOLI
56
BCCP PSEAE
100
49
BCCP ARATH
67
BCCP SOYBN
46
BCCP BACSU
HP0371
56
50
slr0435
CT123
44
aq 1363
75
59
99
PAB1771
VIII
94
PH0834
PH1284
62
ancestral node fission (13) fusion (7)
PYCB METJA
63
V
PYCB METTH
78
SSO2464
90
79
aq 1520
87
aq 1614
83
AF2216
AF2085
SPy1183
55
86
VII
OADA PASMU
77
97
DCOA SALTY
DCOA KLEPN
49
63
OADA VIBCH
48
OAD2 VIBCH
Vng1532G
97
81
BCCP PROMO
96
VIII
BCCP VEIPA
BCCP ACIFE
PYCB PSEAE
100
PYC1 YEAST
59
67
IV
PYC2 YEAST
93
PYCA ASPNI
69
PYCA CAEEL
94
PYCA AEDES
54
90
PYCA DROME
99
84
PYC HUMAN
PYC MOUSE
82
PYCA BACSU
PYCA LACLA
X
84
DUR1 YEAST
PYCA MYCTU
90
53
PYCA MLOTI
IX
TR GIARDIA
10
20
PycB tree
DCOA SALTY
100
99
DCOA KLEPN
100
OADA PASMU
100
OAD2 VIBCH
81
OADA VIBCH
OADA TREPA
58
TR_PROFR
OADA STRPY
PYCB PSEAE
62
aq 1614
71
aq 1520
76
PH0834
PYCB METJA
96
PYCB METTH
100
PYC HUMAN
96
PYC MOUSE
PYCA DROME
100
100
PYCA AEDES
100
PYCA CAEEL
PYC2 YEAST
100
100
PYC1 YEAST
PYCA ASPNI
PYCA MLOTI
81
PYCA BACSU
100
69
PYCA LACLA
TR GIARDIA
PYCA MYCTU
0.1
21
Evolution of the BCCP domain fusions
? The evolution of the BCCP domain included
several domain fusions leading to ACCT-BCCP and
PycB-BCCP domain combinations ? Phylogenetic
trees reconstructed from separate alignments of
BCCP and PycB domains demonstrated that BCCP
domains found in pyruvate carboxylase and
oxaloacetate decarboxylase appear to have evolved
via multiple independent gene fusion and fission
events ? For BCCP and biotin carboxylase (BC)
domains the evidence was not robust enough to
discriminate between the two evolutionary
scenarios. BCCP-BC evolved as a single unit in
eukaryotic acetyl-CoA carboxylases, propionyl-CoA
carboxylases and pyruvate carboxylases from one
ancestor that adapted to a variety of metabolic
scenarios (not shown)
22
ACCT-PycB protein from Giardia ? N-terminal
portion is similar to acetyl-CoA carboxylases ?
C-terminal portion is similar to the pyruvate
carboxylases ? The same domain composition as
in Propionibacterium freudenreichii
BCCP
AccA - Acetyl-CoA carboxylase alpha
AccD - Acetyl-CoA carboxylase beta
Pyruvate/oxaloacetate transcarboxylase
AccAAccD-type carboxyltransferase
-COO-BCCP propionyl-CoA ?? BCCP
methylmalonyl-CoA
PycB-type carboxyltransferase
-COO-BCCP pyruvate ?? BCCP oxaloacetate
23
ACCT-PycB protein from Giardia

• The ATTC-PycB enzyme is the methylmalonyl-CoApyru
  vate transcarboxylase
• AccA-AccD-PycB domain fusion is also found in
  Entamoeba histolytica
• The domain organization of the ACCT-PycB enzyme
  from Giardia cannot be explained by a simple
  fusion of the three subunits of the
  Propionibacterium transcarboxylase
• The methylmalonyl-CoApyruvate transcarboxylase
  in Propionibacterium freudenreichii might be
  involved in balancing gluconeogenesis with fatty
  acid biosynthesis
• Giardia is incapable of fatty acid biosynthesis
  and degradation no corresponding genes have
  been found so far
• If Giardia also has a membrane-bound oxaloacetate
  decarboxylase to generate transmembrane gradient
  of Na (sodium motive force), then its ACCT-PycB
  enzyme might catalyze the conversion of pyruvate
  into oxaloacetate.
• More specific function remains to be established

24
Conclusions
? The ACCT-PycB enzyme from Giardia is
methylmalonyl-CoApyruvate transcarboxylase ?
Sequence comparisons do not always allow for
accurate functional predictions of the
biotin-dependent enzymes because of the
multi-domain organization and a variety of
reactions catalyzed by them ? Functional
assignments must also rely on the subunit
organization, gene content in the particular
genome, and phylogenetic analysis ? The
evolution of the BCCP domain included several
domain fusion events leading to ACCT-BCCP and
PycB-BCCP domain combinations ? PycB-BCCP
fusions in pyruvate carboxylases and oxaloacetate
decarboxylases occurred on several independent
occasions in different prokaryotic lineages
25
Acknowledgements

• Heinrich-Heine University of Düsseldorf
• Katrin Henze
• NCBI/NLM/NIH
• I. King Jordan
• Michael Y. Galperin
• Eugene V. Koonin

26
Delineating domain hierarchies in the Conserved
Domain Database (CDD)

• Exporting of pre-existing domain sets from
  PFAM, SMART and COG databases. Converting their
  alignment models into searchable databases of
  Position Specific Score Matrices (PSSMs).
  Results of pre-calculated CD searches for
  proteins in Entrez are stored in CDART.
• The update process mines the CDART database
  for additional members, which meet the 67
  sequence similarity cutoff. Proteins found in
  model organisms or preferred taxonomic nodes are
  also added even if they show more than 67
  similarity to the existing members.
• Sequence-based clustering methods
  (BLASTCLUST), structure-guided alignments (CN3D),
  phylogenetic trees (CDTree, SEALS), phyletic
  patterns (COGs), contextual information, 3D
  structures, and functional information were used
  to create domain hierarchies. New members are
  added by using PSI-BLAST.

27
HisJ/GlnH family of periplasmic binding proteins
YfhD
HemC Porphobilinogen deaminase
MidA
Y4tE
AtmA
7674449
12725102
7379700
14971614
3860717
10567338
ArtJ
3328806
14022782
HisJ
9947192
GluR0
21290119
GlnH
12231027
SocA
GluRC
BvgS
GtpB
EvgS
CjaA
GluB
ScrB
PEB1
FliY
10