Evolution of Metabolic Pathway 10/03/2007

Reconstruction of metabolic pathways in sequenced genomes Metabolic pathways are the most well-understood pathways in the model organisms, and are relatively conserved across all the three domain of organisms; One goal of computational genomics is to reconstruct the metabolic pathways in all sequenced genomes; An naïve solution to this problem is to use the metabolic pathways in model organisms as templates to reconstruct the corresponding pathways in newly sequenced genomes through othologs identification (COG database, BDBH). However, it turns out that the problem is far more complicated to be fully automated due to a few seasons.

Problems for pathway mapping through orthologs identification The missing gene problem due to non-orthologous gene displacement; Multiple paralogs in a genome; Gene loss/reduction. g1 g2 g3 g4 g5 gi Components in template pathway Orthology mapping Non-orthologous gene displacement or gene loss Mapped components in target genome ???? g’1 g’2 g’3 g’4

Carbohydrate Metabolism Glucose Glycolysis pathway: Encoded in all the organism, except Rickettsia prowazekii, a intracellular parasite. Non-orthologous gene displacement is found in 6 of 10 enzymes in this pathway

Glycolysis pathway Glucokinase: not a essential gene, but at least three analogs are found: Glucose COG0837 Classic in E.coli ADP-dependent New form in P. furious PEP-dependent phosphotransferase A transporter type

Glycolysis pathway Glucose-6-phosphate isomerase(GPI) COG0166 The classical (E. coli) form found in most eubacteria and archaea and in the cytoplasm of the eukaryotic cell. COG2140 Encoded in P. furiosus P. horikoshii and A. fulgidus,

Glycolysis pathway Phosphofructokinase (Pkf) an example of an enzyme where several “missing” enzyme forms have been discovered recently COG0205 An ATP-dependent kinase of unique structure found in bacteria and many eukaryotes. PH1645 ADP-dependent PKF found in P. furiosus and M. jannaschii. APE0012 found in Halobacterium sp., A. fulgidus, M. thermoautotrophicum, A. pernix, and several other archaea

Glycolysis pathway Fructose-1,6-bisphosphate aldolase (PB) COG0191 a metal-independent one (class I) in bacteria and multicellular eukaryotes COG1830 and a metal-dependent one (class II) in archaea, bacteria and yeast These two forms have been known for more than 50 years. Some organisms have both, eg. E. coli.

Glycolysis pathway Phosphoglycerate mutase COG0588 2,3-bisphosphoglycerate-dependent (animal-type) COG0696 2,3-bisphosphoglycerate-independent (plant-type), COG3635 distantly related to the cofactor-independent phosphoglycerate mutase, found in archaea.

Glycolysis pathway Pyruvate kinase COG0469 Irreversible form COG0574 Reversible form, also called phosphoenolpyruvate synthase

Carbohydrate Metabolism Gluconeogenesis pathway With the exception of reactions catalyzed by phosphofructokinase and pyruvate kinase, glycolytic reactions are reversible and function also in gluconeogenesis

Gluconeogenesis pathway Fructose-1,6-bisphosphatase COG0158 found in E. coli, yeast, and human) COG1494 originally described in cyanobacteria BS_fbp encoded in B. subtilis MJ0109 Encod in archaea as M. jannaschii

Carbohydrate Metabolism Phosphoenolpyruvate synthase COG0574 widely present in bacteria, archaea, protists, and plants but missing in animals, where PEP is synthesized from oxaloacetates in a PEP carboxykinase-catalyzed reaction

Carbohydrate Metabolism Phosphoenolpyruvate carboxykinase COG1866 oxaloacetate + ATP <=> phosphoenolpyruvate + ADP + CO2 found in plants, yeast, and many bacteria, potential drug target. COG1274 oxaloacetate + GTP <=> phosphoenolpyruvate + GDP + CO2 found in animals and in a limited number of bacteria.

Carbohydrate Metabolism Entner-Doudoroff pathway and pentose phosphate shunt Found in many organisms but not universal; convert hexoses and pentoses into trioses. Novel enzymes need to be discovered, maybe Important for biofuel studies.

Carbohydrate Metabolism The tricarboxylic acid cycle (TCA or Krebs cycle) The complete TCA cycle is only found in a handful of microorganisms. Most organisms encode only a certain subset of TCA enzymes and, utilize only fragments of it.

The tricarboxylic acid cycle (TCA or Krebs cycle) The pathway is very diverse: cases of non-orthologous gene displacement are detectable for at least five of the nine TCA cycle enzymes.

The tricarboxylic acid cycle (TCA or Krebs cycle) Non-orthologous gene displacement in TCA cycle not related Distantly related Malate dehydrogenase Aconitase Citrate synthase Fumarate hydratase Isocitrate dehydrogenase Succinate dehydrogenase Succinyl-CoA synthetase 2-Ketoglutarate dehydrogenase

The tricarboxylic acid cycle (TCA or Krebs cycle) There are still missing genes in TCA of some genomes Malate dehydrogenase Aconitase Citrate synthase Fumarate hydratase Isocitrate dehydrogenase Succinate dehydrogenase Succinyl-CoA synthetase 2-Ketoglutarate dehydrogenase

Pyrimidine Biosynthesis Has fairly consistent phylogenetic profiles, 3 cases of non-orthologous gene displacement; Carbamoyl phosphate synthase Aspartate carbamoyltransferase The pathway, with the exception of the last 3 steps, is missing in the obligate parasitic bacteria with small genomes: rickettsiae, chlamydiae, spirochetes, and mycoplasmas. Bacteria and archaea with larger genomes encode all or almost all enzymes of pyrimidine biosynthesis Dihydroorotase Dihydroorotate dehydrogenase Orotate phosphoribosyltransferase Orotidine-5′-monophosphate decarboxylase Uridylate kinase Nucleoside diphosphate kinase CTP synthase

Pyrimidine Biosynthesis There appears to be a tendency toward decreasing the genome size by losing genes that have ceased to be essential. The trend toward gene loss is much more pronounced for the initial steps of the pyrimidine biosynthesis pathway than it is for the distal steps. Thus, while depending on the host for the supply of essential nutrients, a parasite preserves at least some metabolic plasticity.

Purine Biosynthesis Has fairly consistent phylogenetic profiles, although cases of non-orthologous gene displacement can be found. Some enzymes are missing in parasitic bacteria with small genomes, namely mycoplasmas, rickettsiae, chlamydiae, spirochetes, Buchnera sp., and H. pylori.

Amino Acid Biosynthesis Aromatic amino acids Biosyntheses of tryptophan The biosynthetic pathways for phenylalanine, tyrosine, and tryptophan in bacteria and eukaryotes share common steps leading from phosphoenolpyruvate and erythrose-4-phosphate to chorismate; Enzymes for most of these steps are encoded also in archaeal genomes;

Biosynthesis of biosyntheses of tryptophan There are three forms of 2-Dehydro-3-deoxy-D-arabino-heptonate 7-phosphate synthase (DAHP). The overlap of the phyletic patterns for the three forms of DAHP exactly corresponds to the the phyletic pattern of the downstream enzyme 3-dehydroquinate synthase.

Biosynthesis of biosyntheses of tryptophan Dehydroquinate dehydratase is found in several genomes that do not encode dehydroquinate synthase, and DAHP, indicating the existence of an alternative, still uncharacterized pathway of dehydroquinate formation in these species The phyletic pattern for shikimate 5-dehydrogenase coincides with the combined pattern of the two forms of 3-dehydroquinate dehydratases:

Biosynthesis of phenylalanine and tyrosine Non-orthologous gene displacement are found for the 3 enzymes led to the syntheses of phenylalanine and tyrosine from chorismate.

A summary on aromatic amino acid biosynthesis Most bacteria and archaea retain the complete set of genes for tryptophan biosynthesis. The exceptions are the obligate archaeal heterotroph P. horikoshii and some obligate bacterial parasites; Enzymes of the tyrosine biosynthesis pathway are encoded in almost as many complete genomes, with the exception of P. abyssi (live in 105ºC). A. aeolicus and four archaeal species encode 3-dehydroquinate dehydratase and all the downstream enzymes but do not encode either DAHP synthase or 3-dehydroquinate synthase: It appears that these organisms produce 3-dehydroquinate via a different mechanism, which does not include DAHP as an intermediate.

Coenzyme Biosynthesis Thiamine Biosynthesis Not well-studied, every enzyme has its own distinct phylogenetic pattern, indicating the abundance of non-orthologous gene displacement cases. There is still ample opportunity for new discoveries in other organisms.    

Coenzyme Biosynthesis Riboflavin Biosynthesis Three of the seven rib genes characterized in E. coli and B. subtilis having no archaeal orthologs; There is an excellent chance of discovering new enzymes of this pathway.

Microbial enzymes as potential drug targets One of the major goals of bacterial pathogen genome sequencing projects is to better understand their peculiarities and to develop new approaches for controlling diseases caused by these organisms. Comparative genomics studies can help to choose drug candidates that are most likely to be effective and least likely to be toxic. In fact, the known targets of all antibiotics are essential for bacterial cell metabolism and not represented in human cells. The “parts list” encoded in the pathogen genomes offers a wide selection of potential drug targets.

Potential targets for broad-spectrum drugs Essential genes for each particular group of bacteria (all bacteria, all Gram-positive bacteria, all mycobacteria, etc.), e.g., Cell wall synthesis pathways Transcription and translation machineries; DNA replication pathways; Purine and pirimidine synthesis pathways Uncharacterized genes whose wide distribution in microbial genomes marks them as being most likely essential.

Potential targets for pathogen-specific drugs Membrane transport systems: Many pathogens rely on the host for the supply of certain essential nutrients, the respective membrane transport systems can be valid targets for drug intervention, e.g., The still uncharacterized biotin transport system appears to be the only means of biotin acquisition for several pathogens, such as S. pyogenes, R. prowazekii, C. trachomatis, and T. pallidum . Surface proteins of bacteria as drug targets Host interaction factors that are essential for establishing parasitic or infectious state. Analogous proteins in certain pathogens in a different form than present in humans. Detailed analysis of non-orthologous displacement cases has led to suggestions that alternative forms of essential enzymes could be used as drug targets

Methods for drug target selection Phylogenetic profiles can be used to identify genes that are essential to all (or most) pathogenic microorganisms in a chosen group, as well as those that are specific for a particular organism. The products of the former set are attractive targets for broad-spectrum antibiotics, whereas the unique proteins offer an opportunity to design specific antibiotics, which would specifically target a narrow group of bacteria or even one particular pathogen. Pathogens Non-pathogens and eukaryotes COG1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 COG2 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 COG3 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 COG4 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 COG5 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 COG6 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 COG7 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Examples of pathogen-specific drug targets Enzymes with limited phylogenetic distribution Human pathogens that depend on these enzymes ATP/ADP translocase, bacterial/plant type R. prowazekii, C. trachomatis, C. pneumoniae 3-Dehydroquinate dehydratase, class II C. jejuni, H. influenzae, H. pylori, P. aeruginosa, V. cholerae DhnA-type fructose-1,6-bisphosphate aldolase C. trachomatis, C. pneumoniae Lysyl-tRNA synthetase, class I B. burgdorferi, R. prowazekii, T. pallidum, Na+-translocating NADH: ubiquinone oxidoreductase C. trachomatis, C. pneumoniae, Cl. perfringens, T. denticola Na+-translocating oxalo-acetate decarboxylase S. pyogenes, T. pallidum, Orotidine 5′-phosphate decarboxylase M. leprae, M. tuberculosis Pyridoxine biosynthesis enzymes PDX1, PDX2 Bacillus anthracis, H. influenzae, L. monocytogenes, M. leprae, M. tuberculosis, S. pneumoniae Cofactor-independent phosphoglycerate mutase C. jejuni, H. pylori, M. genitalium, P. aeruginosa, V. cholerae

Conclusions Metabolic pathways are very subject to non-orthologous gene displacement; If an organism encodes a significant fraction of the enzymes for a particular pathway, it is likely that a missing enzyme may be sought for among uncharacterized orthologous sets with inverted phylogenetic profiles; Conversely, there are cases where most of the enzymes of a given pathway are missing in an organism but one or two still stay around, most likely, these are cases of exaptation; Identification of potential targets for antibacterial drugs is a natural task for comparative genomics and will likely remain one of its most important practical applications for years to come.