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The functions of most proteins are yet to be determined. The function of an enzyme is often defined by its interacting partners, including its substrate and product, and its role in larger metabolic networks. Here, we describe a computational method that predicts the functions of orphan enzymes by organizing them into a linear metabolic pathway. Given candidate enzyme and metabolite pathway members, this aim is achieved by finding those pathways that satisfy structural and network restraints implied by varied input information, including that from virtual screening, chemoinformatics, genomic context analysis, and ligand -binding experiments. We demonstrate this integrative pathway mapping method by predicting the L-gulonate catabolic pathway in Haemophilus influenzae Rd KW20. The prediction was subsequently validated experimentally by enzymology, crystallography, and metabolomics. Integrative pathway mapping by satisfaction of structural and network restraints is extensible to molecular networks in general and thus formally bridges the gap between structural biology and systems biology.

Using a large-scale "genomic enzymology" approach, we (i) assigned novel ATP-dependent four-carbon acid sugar kinase functions to members of the DUF1537 protein family (domain of unknown function; Pfam families PF07005 and PF17042) and (ii) discovered novel catabolic pathways for d-threonate, l-threonate, and d-erythronate. The experimentally determined ligand specificities of several solute binding proteins (SBPs) for TRAP (tripartite ATP-independent permease) transporters for four-carbon acids, including d-erythronate and l-erythronate, were used to constrain the substrates for the catabolic pathways that degrade the SBP ligands to intermediates in central carbon metabolism. Sequence similarity networks and genome neighborhood networks were used to identify the enzyme components of the pathways. Conserved genome neighborhoods encoded SBPs as well as permease components of the TRAP transporters, members of the DUF1537 family, and a member of the 4-hydroxy-l-threonine 4-phosphate dehydrogenase (PdxA) oxidative decarboxylase, class II aldolase, or ribulose 1,5-bisphosphate carboxylase/oxygenase, large subunit (RuBisCO) superfamily. Because the characterized substrates of members of the PdxA, class II aldolase, and RuBisCO superfamilies are phosphorylated, we postulated that the members of the DUF1537 family are novel ATP-dependent kinases that participate in catabolic pathways for four-carbon acid sugars. We determined that (i) the DUF1537/PdxA pair participates in a pathway for the conversion of d-threonate to dihydroxyacetone phosphate and CO2 and (ii) the DUF1537/class II aldolase pair participates in pathways for the conversion of d-erythronate and l-threonate (epimers at carbon-3) to dihydroxyacetone phosphate and CO2 The physiological importance of these pathways was demonstrated in vivo by phenotypic and genetic analyses.

Metabolic pathways in eubacteria and archaea often are encoded by operons and/or gene clusters (genome neighborhoods) that provide important clues for assignment of both enzyme functions and metabolic pathways. We describe a bioinformatic approach (genome neighborhood network; GNN) that enables large scale prediction of the in vitro enzymatic activities and in vivo physiological functions (metabolic pathways) of uncharacterized enzymes in protein families. We demonstrate the utility of the GNN approach by predicting in vitro activities and in vivo functions in the proline racemase superfamily (PRS; InterPro IPR008794). The predictions were verified by measuring in vitro activities for 51 proteins in 12 families in the PRS that represent ∼85% of the sequences; in vitro activities of pathway enzymes, carbon/nitrogen source phenotypes, and/or transcriptomic studies confirmed the predicted pathways. The synergistic use of sequence similarity networks3 and GNNs will facilitate the discovery of the components of novel, uncharacterized metabolic pathways in sequenced genomes.

Assigning valid functions to proteins identified in genome projects is challenging: overprediction and database annotation errors are the principal concerns (1). We and others (2) are developing computation-guided strategies for [...]

The biosynthesis of cysteine in bacteria and plants is carried out by a two-step pathway, catalyzed by serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS; O-acetylserine [thiol] lyase). The aerobic form of OASS forms a tight bienzyme complex with SAT in vivo, termed cysteine synthase. We have determined the crystal structure of OASS in complex with a C-terminal peptide of SAT required for bienzyme complex formation. The binding site of the peptide is at the active site of OASS, and its C-terminal carboxyl group occupies the same anion binding pocket as the [alpha]-carboxylate of the O-acetylserine substrate of OASS. These results explain the partial inhibition of OASS by SAT on complex formation as well as the competitive dissociation of the complex by O-acetylserine.

The X-ray crystal structures of homoprotocatechuate 2,3-dioxygenases isolated from Arthrobacter globiformis and Brevibacterium fuscum have been determined to high resolution. These enzymes exhibit 83% sequence identity, yet their activities depend on different transition metals, [Mn.sup.2+] and [Fe.sup.2+], respectively. The structures allow the origins of metal ion selectivity and aspects of the molecular mechanism to be examined in detail. The homotetrameric enzymes belong to the type I family of extradiol dioxygenases (vicinal oxygen chelate super-family); each monomer has four [beta][alpha][beta][beta][beta] modules forming two structurally homologous N-terminal and C-terminal barrel-shaped domains. The active-site metal is located in the C-terminal barrel and is ligated by two equatorial ligands, H[214.sup.NE1] and E[267.sup.OE1]; one axial ligand, H[155.sup.NE1]; and two to three water molecules. The first and second coordination spheres of these enzymes are virtually identical (root mean square difference over all atoms, 0.19 [Angstrom]), suggesting that the metal selectivity must be due to changes at a significant distance from the metal and/or changes that occur during folding. The substrate (2,3-dihydroxyphenylacetate [HPCA]) chelates the metal asymmetrically at sites trans to the two imidazole ligands and interacts with a unique, mobile C-terminal loop. The loop closes over the bound substrate, presumably to seal the active site as the oxygen activation process commences. An 'open' coordination site trans to E267 is the likely binding site for [O.sup.2]. The geometry of the enzyme-substrate complexes suggests that if a transiently formed metal-superoxide complex attacks the substrate without dissociation from the metal, it must do so at the C-3 position. Second-sphere active-site residues that are positioned to interact with the HPCA and/or bound [O.sup.2] during catalysis are identified and discussed in the context of current mechanistic hypotheses.

The results of the analysis on the nonheme ferric iron intradiol dioxygenase protocatechuate 3,4-dioxygenase is reviewed. It is found out that the catechol dioxygenases allow a wide variety of bacteria to use aromatic compounds as carbon sources by catalyzing the key ring-opening step.

Results show that protocatechuate 3,4-dioxygenases possess three sites for substrate binding. Data also indicate that the overall tertiary and quaternary structures of the enzyme from Acinetobacter Strain ADP1 and Pseudomonas putida are similar.

The kinetic characterization of bifunctional aminoglycoside acetyltransferase-Ib variant (AAC(6')-Ib-cr) and its wild-type parent enzyme along with the three-dimensional structure of the wild-type enzyme is described. It highlights the adaptive potential of AACs driven by the selective pressures of fluoroquinolone use.

The study of the interaction of Mycobacterium tuberculosis enoyl-ACP reductase with 1,2,isonicotinoyl-NAD(P) [INH-NADP] adducts is being discussed. The analysis has been extremely helpful, as it gives a complete analysis of the mechanism of the action and resistance to isoniazid.

The studies on the basis of kinetic properties, pH dependence, and structural information of SSAT in Escherichia coli reveal an acid/base-assisted reaction catalyzed by spermidine/spermine acetyltransferase (SSAT), involving a ternary complex.

The three-dimensional structure of the first member of the pentapeptide repeat protein (PRP) family is determined for the fluoroquinolene resistance protein (MfpA) from Mycobacterium tuberculosis. The structure shows that the pentapeptide repeats encode the folding of a novel right-handed quadrilateral beta-helix, MfpA binds to DNA gyrase and inhibits its activity and the rod-shaped, dimeric protein displays remarkable similarity in size, shape and electrostatics to DNA.

Mutant enzymes that exhibit less than 0.1% wild-type activity are used to explore the role of Tyr408. The X-ray crystal structures of the mutants and their PCA complexes show that the new shorter residues in the 408 position cannot ligate the iron and instead interact with iron through solvents.

The X-ray crystal structure of Haemophilus influenzae serine acetylytransferase (SAT) in complexes with CoA and its cysteine feedback inhibitor is determined. The overall polypeptide chain fold of the hexameric enzyme and the location of its active site are defined.

Using a large-scale "genomic enzymology" approach, we (i) assigned novel ATP-dependent four-carbon acid sugar kinase functions to members of the DUF1537 protein family (domain of unknown function; Pfam families PF07005 and PF17042) and (ii) discovered novel catabolic pathways for D-threonate, L-threonate, and D-erythronate. The experimentally determined ligand specificities of several solute binding proteins (SBPs) for TRAP (tripartite ATP-independent permease) transporters for four-carbon acids, including D-erythronate and L-erythronate, were used to constrain the substrates for the catabolic pathways that degrade the SBP ligands to intermediates in central carbon metabolism. Sequence similarity networks and genome neighborhood networks were used to identify the enzyme components of the pathways. Conserved genome neighborhoods encoded SBPs as well as permease components of the TRAP transporters, members of the DUF1537 family, and a member of the 4-hydroxy-L-threonine 4-phosphate dehydrogenase (PdxA) oxidative decarboxylase, class II aldolase, or ribulose 1,5-bisphosphate carboxylase/oxygenase, large subunit (RuBisCO) superfamily. Because the characterized substrates of members of the PdxA, class II aldolase, and RuBisCO superfamilies are phosphorylated, we postulated that the members of the DUF1537 family are novel ATP-dependent kinases that participate in catabolic pathways for four-carbon acid sugars. We determined that (i) the DUF1537/PdxA pair participates in a pathway for the conversion of D-threonate to dihydroxyacetone phosphate and CO2 and (ii) the DUF1537/class II aldolase pair participates in pathways for the conversion of D-erythronate and L-threonate (epimers at carbon-3) to dihydroxyacetone phosphate and CO2. The physiological importance of these pathways was demonstrated in vivo by phenotypic and genetic analyses. [ABSTRACT FROM AUTHOR]