Subsystem: FMN/FAD Biosynthesis Andrei Osterman1,2 and Dimitry Rodionov3 1 The Burnham Institute, 2 FIG, Institute for Information Transmission ProblemsM oscow, Russia
Introduction and Subsystem Notes Riboflavin (vitamin B2) is an ultimate precursor in the biosynthesis of the universal and indispensable flavonoid red-ox cofactors: flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Many aspects of riboflavin/FMN/FAD biosynthesis are conserved across all taxons (for a review see [1]). The most significant variation within this subsystem occurs in archaea, where some homologous and non-homologous forms of previously known enzymes have already been characterized [2-4], but a number of enzymatic steps are still associated with missing genes. In a recent study, an alternative two-step transformation (via a hypothesized GTP cyclohydrolase III and nucleotide triphosphate pyrophosphatase) was proposed [2] instead of the established onestep reaction of GTP-cyclohydrolase II characteristic of bacteria, fungi and plants. Another remarkable variation is the flipped order of the pyrimidine deaminase and reductase steps in eukaryotes and archaea, compared to the standard bacterial biosynthetic route. Both, fungi and archaea contain a monofunctional reductase, homologous to a corresponding domain of the bifunctional protein (encoded by ribD gene) characteristic of most bacteria. A monofunctional and non-orthologous eukaryotic deaminase (Pyr_Da) was identified and characterized, while its archaeal counterpart remains a missing gene. Analysis of chromosomal gene clustering in archaea reveals a strong candidate gene for this functional role. Orthologs of this gene (e.g., MJ0699 in M. jannaschii) are both distantly related to guanosine deaminase and largely conserved in archaea,
S ubsystem: FMN/FAD Biosynthesis
Introduction and Subsystem Notes (continued)
A discovery of a riboswitch-type regulatory mechanism in bacterial riboflavin biosynthesis [5] enabled a prediction and experimental verification [6] of a riboflavin transporter (gene ypaA of B.subtilis), which appears to be conserved in a number of gram-positive bacteria. An extensive comparative genomics analysis of chromosomal clustering and regulatory sites shared by riboflavin biosynthetic genes allowed to make a number of conjectures, eg a prediction of novel riboflavin transporters [7]. Pyrimidine phosphatase (PyrP), a necessary step of the pathway implicated by the biochemical data, remains a “globally missing” gene. A semi-automated identification and analysis of functional variants of the subsystem was performed across >260 complete or almost complete genomes in SEEE (Y.Ye et al, 2005, published in ISMB’05). We have observed a limited number of variants, and some of them are illustrated in the following slides (also see Subsystem Notes in SEED). An overwhelming majority of bacteria have an absolutely conserved complete de novo biosynthesis, and at least some of them (but not all, eg E.coli) are also capable to uptake salvage exogenous riboflavin (see above). Relatively small number of pathogens as well as multicellular eukaryotes are completely dependent on utilization of exogenous riboflavin. They contain only two enzymes of the “universal pathway, FK and FMNAT. The latter enzyme (conserve and essential in most bacteria) appears to be a promising antiinfective target [8], due to a nonorthologous replacement in all eukaryotes (FMNAT2). A human enzyme was experimentally verified and characterized by F.Mseeh, A.Osterman et al (unpublished data). Archaeal forms of both RK and FMNAT are still unknown.
1. Functional Roles, Abbreviations, Subsets and Alternative Forms of Enzymes Alternative forms
S ubsystem: FMN/FAD Biosynthesis
Subsets of roles
*Pyr_D De novo biosynthesis
*RS A
*FMNAT
Universal pathway
Subsystem spreadsheet a fragment of the SEED display with selected examples
S ubsystem: FMN/FAD Biosynthesis
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? ? ? ?
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?
?
?
M atching colors highlight genes that occur close to each other on the chromosome. Genes (proteins) assigned with respective functional roles are shown by unique FIG IDs. Alternative forms are indicated by additional numbers, dash-separated.”M issing genes” are indicated by “?”. Some of the examples are further illustrated by projection on a subsystem diagram.
FMN/FAD Biosynthesis RIBOFLAVIN SALVAGE B2 (TRANSPORT)
Subsystem diagram Example E.coli (variant 1) Comments: No transport of exogenous riboflavin in wt strain. All genes of the pathway are essential (see Gerdes et al., 2003)
ARCHAEA LBRANCH hypothetical
NADPH
Pyr_R
Pyr_D
III
Pyr_P
IV
Pyr_R
genes unknown
V
RSA
B2
RK
PP i
FM NAT
FMN
FAD FM NAT2
\ UNIVERSAL FM N/FAD PATHWAY DHBPS
VIII variant in archaea and fungi
GTP Guanosine triphosphate I 2,5-Diamino-6-ribosylamino-4(3H)pyrimidinone 5'-phosphate II 5-Amino-6-ribosylamino-2,4(1H,3H)pyrimidinedione 5'-phosphate
VI
GTPCH2
H2 O
DM RS
ADP
VII
H2 O
PP i, formate
formate
GTPCH3
absent
ATP
Pyr_D (e or a)
I
IX
YpaA
Pi
NH3
H2 O
H2 O
?
H2 O
NADP
NH3
NTPPP
essential dispensable
ATP
II
Pi
Functional role abbreviations (in boxes) are as in Panel 1. Color coding scheme:
III
H2 O
GTP RIBOFLAVIN DE NOVO BIOSYNTHESIS Pentose Phosphate Cycle
Purine Nucleotide Biosysnthesis
?
5-Amino-6-ribitylamino-2,4(1H,3H)pyrimidinedione 5'-phosphate IV 5-Amino-6-ribitylamino-2,4(1H,3H)pyrimidinedione V 6,7-Dimethyl-8-ribityl-lumazine VI 2,5-diamino-6-ribosylamino-4(3H)pyrimidinone 5-triphosphate VI Ribulose 5-phosphate VII L-3,4-Dihydroxy-2-butanone 4phosphate B2 Riboflavin FMN Flavin Mononucleotide FAD Flavin Adenine Dinucleotide VIII 2,5-diamino-6-ribitylamino-4(3H)pyrimidinone 5-phosphate IX 2,5-diamino-6-ribosylamino-4(3H)pyrimidinone 5-triphosphate
FMN/FAD Biosynthesis
Subsystem diagram Example: B.subtilis (variant 11)
RIBOFLAVIN SALVAGE B2 (TRANSPORT)
Comments: ypaA gene was confirmed as ribofavin transporter. Only last two genes are essential (see Kobayashi et al., 2003). Same pattern is observed in S.aureus, S.pneumoniae and many other gram-positive bacteria ARCHAEA LBRANCH hypothetical
NADPH
Pyr_R
Pyr_D
III
Pyr_P
IV
Pyr_R
genes unknown
V
RSA
B2
RK
PP i
FM NAT
FMN
FAD FM NAT2
\ UNIVERSAL FM N/FAD PATHWAY DHBPS
VIII variant in archaea and fungi
GTP Guanosine triphosphate I 2,5-Diamino-6-ribosylamino-4(3H)pyrimidinone 5'-phosphate II 5-Amino-6-ribosylamino-2,4(1H,3H)pyrimidinedione 5'-phosphate
VI
GTPCH2
H2 O
DM RS
ADP
VII
H2 O
PP i, formate
formate
GTPCH3
absent
ATP
Pyr_D (e or a)
I
IX
YpaA
Pi
NH3
H2 O
H2 O
?
H2 O
NADP
NH3
NTPPP
essential dispensable
ATP
II
Pi
Functional role abbreviations (in boxes) are as in Panel 1. Color coding scheme:
H2 O
GTP
III
RIBOFLAVIN DE NOVO BIOSYNTHESIS Pentose Phosphate Cycle
Purine Nucleotide Biosysnthesis
?? ?
5-Amino-6-ribitylamino-2,4(1H,3H)pyrimidinedione 5'-phosphate IV 5-Amino-6-ribitylamino-2,4(1H,3H)pyrimidinedione V 6,7-Dimethyl-8-ribityl-lumazine VI 2,5-diamino-6-ribosylamino-4(3H)pyrimidinone 5-triphosphate VI Ribulose 5-phosphate VII L-3,4-Dihydroxy-2-butanone 4phosphate B2 Riboflavin FMN Flavin Mononucleotide FAD Flavin Adenine Dinucleotide VIII 2,5-diamino-6-ribitylamino-4(3H)pyrimidinone 5-phosphate IX 2,5-diamino-6-ribosylamino-4(3H)pyrimidinone 5-triphosphate
FMN/FAD Biosynthesis
Subsystem diagram Example: L.innocua (variant 21) Comments: No de novo pathways . A transporter (YpaA homolog) should be essential for this functional variant. On the other hand, FK/FMNAT homologs may behave as nonessential genes due to redundancy. ARCHAEA LBRANCH hypothetical
NADPH
Pyr_R
Pyr_D
Pyr_D (e or a)
I
IX
H2 O
Pyr_R PP i, formate
GTPCH2 formate
GTPCH3 H2 O
genes unknown
Pyr_P NH3
H2 O
H2 O
III
ATP
Pi
H2 O
NADP
NH3
NTPPP
YpaA ATP
II
Pi
RIBOFLAVIN SALVAGE B2 (TRANSPORT)
VIII variant in archaea and fungi
IV
DM RS
V
RSA
B2
ADP
RK
FM NAT
FMN
FAD FM NAT2
VII
\ UNIVERSAL FM N/FAD PATHWAY DHBPS GTP Guanosine triphosphate I 2,5-Diamino-6-ribosylamino-4(3H)pyrimidinone 5'-phosphate II 5-Amino-6-ribosylamino-2,4(1H,3H)pyrimidinedione 5'-phosphate
VI
H2 O
GTP
III
RIBOFLAVIN DE NOVO BIOSYNTHESIS
Purine Nucleotide Biosysnthesis
PP i
Pentose Phosphate Cycle
5-Amino-6-ribitylamino-2,4(1H,3H)pyrimidinedione 5'-phosphate IV 5-Amino-6-ribitylamino-2,4(1H,3H)pyrimidinedione V 6,7-Dimethyl-8-ribityl-lumazine VI 2,5-diamino-6-ribosylamino-4(3H)pyrimidinone 5-triphosphate VI Ribulose 5-phosphate VII L-3,4-Dihydroxy-2-butanone 4phosphate B2 Riboflavin FMN Flavin Mononucleotide FAD Flavin Adenine Dinucleotide VIII 2,5-diamino-6-ribitylamino-4(3H)pyrimidinone 5-phosphate IX 2,5-diamino-6-ribosylamino-4(3H)pyrimidinone 5-triphosphate
S ubsystem: FMN/FAD Biosynthesis
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