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1.  Mechanism of Dihydroneopterin Aldolase: The Functional Roles of the Conserved Active Site Glutamate and Lysine Residues† 
Biochemistry  2006;45(51):15232-15239.
Dihydroneopterin aldolase (DHNA) catalyzes the conversion of 7,8-dihydroneopterin (DHNP) to 6-hydroxymethyl-7,8-dihydropterin (HP) in the folate biosynthetic pathway. There are four conserved active site residues at the active site, E22, Y54, E74, and K100 in Staphylococcus aureus DHNA (SaDHNA), corresponding to E21, Y53, E73, and K98 in Escherichia coli DHNA (EcDHNA). The functional roles of the conserved glutamate and lysine residues have been investigated by site-directed mutagenesis in this work. E22 and E74 of SaDHNA and E21, E73, and K98 of EcDHNA were replaced by alanine. K100 of SaDHNA was replaced by alanine and glutamine. The mutant proteins were characterized by equilibrium binding, stopped-flow binding, and steady-state kinetic analyses. For SaDHNA, none of the mutations except E74A caused dramatic changes in the affinities of the enzyme for the substrate or product analogues or the rate constants. The Kd values for SaE74A were estimated to be >3000 μM, suggesting that the Kd values of the mutant is at least 100 times those of the wild-type enzyme. For EcDHNA, the E73A mutation caused increases in the Kd values for the substrate or product analogues neopterin (MP), monapterin (NP), and 6-hydroxypterin (HPO) by factors of 340, 160, and 5600, respectively, relative to those of the wild-type enzyme. The K98A mutation caused increases in the Kd values for NP, MP, and HPO by factors of 14, 3.6, and 230, respectively. The E21A mutation caused increases in the Kd values for NP and HPO by factors of 2.2 and 42, respectively, but a decrease in the Kd value for MP by a factor of 3.3. The E22 (E21) and K100 (K98) mutations caused decreases in the kcat values by factors of 1.3×104 to 2×104. The E74 (E73) mutation caused decreases in the kcat values by factors of ~10. The results suggested that E74 of SaDHNA and E73 of EcDHNA are important for substrate binding, but their roles in catalysis are minor. In contrast, E22 and K100 of SaDHNA are important for catalysis, but their roles in substrate binding are minor. On the other hand, E21 and K98 of EcDHNA are important for both substrate binding and catalysis.
PMCID: PMC3018710  PMID: 17176045
2.  An atypical orthologue of 6-pyruvoyltetrahydropterin synthase can provide the missing link in the folate biosynthesis pathway of malaria parasites 
Molecular Microbiology  2007;67(3):609-618.
Folate metabolism in malaria parasites is a long-standing, clinical target for chemotherapy and prophylaxis. However, despite determination of the complete genome sequence of the lethal species Plasmodium falciparum, the pathway of de novo folate biosynthesis remains incomplete, as no candidate gene for dihydroneopterin aldolase (DHNA) could be identified. This enzyme catalyses the third step in the well-characterized pathway of plants, bacteria, and those eukaryotic microorganisms capable of synthesizing their own folate. Utilizing bioinformatics searches based on both primary and higher protein structures, together with biochemical assays, we demonstrate that P. falciparum cell extracts lack detectable DHNA activity, but that the parasite possesses an unusual orthologue of 6-pyruvoyltetrahydropterin synthase (PTPS), which simultaneously gives rise to two products in comparable amounts, the predominant of which is 6-hydroxymethyl-7,8-dihydropterin, the substrate for the fourth step in folate biosynthesis (catalysed by 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase; PPPK). This can provide a bypass for the missing DHNA activity and thus a means of completing the biosynthetic pathway from GTP to dihydrofolate. Supported by site-directed mutagenesis experiments, we ascribe the novel catalytic activity of the malarial PTPS to a Cys to Glu change at its active site relative to all previously characterized PTPS molecules, including that of the human host.
PMCID: PMC2229834  PMID: 18093090
3.  One substrate, five products: reactions catalyzed by the dihydroneopterin aldolase from Mycobacterium tuberculosis 
Journal of the American Chemical Society  2012;134(48):19758-19771.
Tetrahydrofolate cofactors are required for one carbon transfer reactions involved in the synthesis of purines, amino acids, and thymidine. Inhibition of tetrahydrofolate biosynthesis is a powerful therapeutic strategy in the treatment of several diseases, and the possibility of using antifolates to inhibit enzymes from Mycobacterium tuberculosis has been explored. This work focuses on the study of the first enzyme in tetrahydrofolate biosynthesis that is unique to bacteria, dihydroneopterin aldolase (MtDHNA). This enzyme requires no metals or cofactors, and does not form a protein mediated Schiff base with the substrate, unlike most aldolases. Here, we were able to demonstrate that the reaction catalyzed by MtDHNA generates three different pterin products, one of which is not produced by other wild type DHNAs. The enzyme-substrate complex partitions 51% in the first turnover to form the aldolase product, 24% to the epimerase product, and 25% to the oxygenase product. The aldolase reaction is strongly pH-dependent, and apparent pKa values were obtained for the first time for this class of enzyme. Furthermore, chemistry is rate-limiting for the aldolase reaction, and the analysis of solvent kinetic isotope effects in steady-state and pre-steady-state conditions, combined with proton inventory studies revealed that two protons and a likely solvent contribution are involved in formation and breakage of a common intermediate. This study provides information about the plasticity required from a catalyst that possesses high substrate specificity while being capable of utilizing two distinct epimers with the same efficiency to generate five distinct products.
PMCID: PMC3530388  PMID: 23150985
Folate biosynthesis; Aldehyde-Lyases; 7,8-dihydroneopterin; Tuberculosis; Kinetics; Solvent Kinetic Isotope Effects; Nuclear Magnetic Resonance; Oxygen chemistry; Oxygenases
4.  Characterization of the Saccharomyces cerevisiae Fol1 Protein: Starvation for C1 Carrier Induces Pseudohyphal Growth 
Molecular Biology of the Cell  2004;15(8):3811-3828.
Tetrahydrofolate (vitamin B9) and its folate derivatives are essential cofactors in one-carbon (C1) transfer reactions and absolutely required for the synthesis of a variety of different compounds including methionine and purines. Most plants, microbial eukaryotes, and prokaryotes synthesize folate de novo. We have characterized an important enzyme in this pathway, the Saccharomyces cerevisiae FOL1 gene. Expression of the budding yeast gene FOL1 in Escherichia coli identified the folate biosynthetic enzyme activities dihydroneopterin aldolase (DHNA), 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase (HPPK), and dihydropteroate synthase (DHPS). All three enzyme activities were also detected in wild-type yeast strains, whereas fol1Δ deletion strains only showed background activities, thus demonstrating that Fol1p catalyzes three sequential steps of the tetrahydrofolate biosynthetic pathway and thus is the central enzyme of this pathway, which starting from GTP consists of seven enzymatic reactions in total. Fol1p is exclusively localized to mitochondria as shown by fluorescence microscopy and immune electronmicroscopy. FOL1 is an essential gene and the nongrowth phenotype of the fol1 deletion leads to a recessive auxotrophy for folinic acid (5′-formyltetrahydrofolate). Growth of the fol1Δ deletion strain on folinic acid–supplemented rich media induced a dimorphic switch with haploid invasive and filamentous pseudohyphal growth in the presence of glucose and ammonium, which are known suppressors of filamentous and invasive growth. The invasive growth phenotype induced by the depletion of C1 carrier is dependent on the transcription factor Ste12p and the flocullin/adhesin Flo11p, whereas the filamentation phenotype is independent of Ste12p, Tec1p, Phd1p, and Flo11p, suggesting other signaling pathways as well as other adhesion proteins.
PMCID: PMC491839  PMID: 15169867
5.  A bifunctional protein in the folate biosynthetic pathway of Streptococcus pneumoniae with dihydroneopterin aldolase and hydroxymethyldihydropterin pyrophosphokinase activities. 
Journal of Bacteriology  1993;175(8):2214-2220.
A protein encoded by sulD, one of four genes in a previously cloned folate biosynthetic operon of Streptococcus pneumoniae, had been shown to harbor 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase activity. This SulD protein was purified and shown now to harbor also dihydroneopterin aldolase activity. The bifunctional protein therefore catalyzes two successive steps in folate biosynthesis. The aldolase activity can be ascribed to the N-terminal domain of the SulD polypeptide, and the pyrophosphokinase activity can be ascribed to the C-terminal domain. Homologs of the dihydroneopterin aldolase domain were identified in other species, in one of which the domain was encoded as a separate polypeptide. The native SulD protein is a trimer or tetramer of a 31-kDa subunit, and it dissociated reversibly after purification. Dihydroneopterin aldolase activity required the multimeric protein, whereas pyrophosphokinase was expressed by the monomeric form. With purified SulD, the amount of 6-hydroxymethyl-7,8-dihydropterin product formed by the aldolase was proportional to the fourth power of the enzyme concentration, as expected for a reversibly dissociating tetramer. By identifying the gene encoding dihydroneopterin aldolase, this work extends our understanding of the molecular basis of the folate biosynthetic system common to many organisms.
PMCID: PMC204506  PMID: 8385663
6.  Characterization of Escherichia coli men Mutants Defective in Conversion of o-Succinylbenzoate to 1,4-Dihydroxy-2-Naphthoate 
Journal of Bacteriology  1982;152(3):1132-1137.
Four independent menaquinone (vitamin K2)-deficient mutants of Escherichia coli, blocked in the conversion of o-succinylbenzoate (OSB) to 1,4-dihydroxy-2-naphthoate (DHNA), were found to represent two distinct classes. Enzymatic complementation was observed when a cell-free extract of one mutant was mixed with extracts of any of the remaining three mutants. The missing enzymes in the two classes were identified by in vitro complementation with preparations of OSB-coenzyme A (CoA) synthetase or DHNA synthase isolated from Mycobacterium phlei. Mutants lacking DHNA synthase (and therefore complementing with M. phlei DHNA synthase) were designated menB, and the mutant lacking OSB-CoA synthetase (and therefore complementing with M. phlei OSB-CoA synthetase) was designated menE. The menB mutants produced only the spirodilactone form of OSB when extracts were incubated with [2,3-14C2]OSB, ATP, and CoA; the OSB was unchanged on incubation with an extract from the menE mutant under these conditions. Experiments with strains lysogenized by a λ men transducing phage (λG68) and transduction studies with phage P1 indicated that the menB and menE genes form part of a cluster of four genes, controlling the early steps in menaquinone biosynthesis, located at 48.5 min in the E. coli linkage map. Evidence was obtained for the clockwise gene order gyrA....menC- 0000100000 0000110000 0011111000 0000111000 0011111000 0001110000 0000110101 0001111111 0001100000 0000100000 0001101100 0011111000 0011000000 0011000000 0111000111 0111101110 -B-D, where the asterisk denotes the uncertain position of menE relative to menC and menB. The transducing phage (λG68) contained functional menB, menC, and menE genes, but only part of the menD gene, and it was designated λ menCB(D).
PMCID: PMC221619  PMID: 6754698
7.  6-Pyruvoyltetrahydropterin Synthase Paralogs Replace the Folate Synthesis Enzyme Dihydroneopterin Aldolase in Diverse Bacteria▿ †  
Journal of Bacteriology  2009;191(13):4158-4165.
Dihydroneopterin aldolase (FolB) catalyzes conversion of dihydroneopterin to 6-hydroxymethyldihydropterin (HMDHP) in the classical folate biosynthesis pathway. However, folB genes are missing from the genomes of certain bacteria from the phyla Chloroflexi, Acidobacteria, Firmicutes, Planctomycetes, and Spirochaetes. Almost all of these folB-deficient genomes contain an unusual paralog of the tetrahydrobiopterin synthesis enzyme 6-pyruvoyltetrahydropterin synthase (PTPS) in which a glutamate residue replaces or accompanies the catalytic cysteine. A similar PTPS paralog from the malaria parasite Plasmodium falciparum is known to form HMDHP from dihydroneopterin triphosphate in vitro and has been proposed to provide a bypass to the FolB step in vivo. Bacterial genes encoding PTPS-like proteins with active-site glutamate, cysteine, or both residues were accordingly tested together with the P. falciparum gene for complementation of the Escherichia coli folB mutation. The P. falciparum sequence and bacterial sequences with glutamate or glutamate plus cysteine were active; those with cysteine alone were not. These results demonstrate that PTPS paralogs with an active-site glutamate (designated PTPS-III proteins) can functionally replace FolB in vivo. Recombinant bacterial PTPS-III proteins, like the P. falciparum enzyme, mediated conversion of dihydroneopterin triphosphate to HMDHP, but other PTPS proteins did not. Neither PTPS-III nor other PTPS proteins exhibited significant dihydroneopterin aldolase activity. Phylogenetic analysis indicated that PTPS-III proteins may have arisen independently in various PTPS lineages. Consistent with this possibility, merely introducing a glutamate residue into the active site of a PTPS protein conferred incipient activity in the growth complementation assay, and replacing glutamate with alanine in a PTPS-III protein abolished complementation.
PMCID: PMC2698474  PMID: 19395485
8.  Menaquinone (Vitamin K2) Biosynthesis: Localization and Characterization of the menA Gene from Escherichia coli 
Journal of Bacteriology  1998;180(10):2782-2787.
A key reaction in the biosynthesis of menaquinone involves the conversion of the soluble bicyclic naphthalenoid compound 1,4-dihydroxy-2-naphthoic acid (DHNA) to the membrane-bound demethylmenaquinone. The enzyme catalyzing this reaction, DHNA-octaprenyltransferase, attaches a 40-carbon side chain to DHNA. The menA gene encoding this enzyme has been cloned and localized to a 2.0-kb region of the Escherichia coli genome between cytR and glpK. DNA sequence analysis of the cloned insert revealed a 308-codon open reading frame (ORF), which by deletion analyses was shown to restore anaerobic growth of a menA mutant. Reverse-phase high-performance liquid chromatography analysis of quinones extracted from the orf-complemented cells independently confirmed the restoration of menaquinone biosynthesis, and similarly, analyses of isolated cell membranes for DHNA octaprenyltransferase activity confirmed the introduction of the menA product into the orf-complemented menA mutant. The validity of an ORF-associated putative promoter sequence was confirmed by primer extension analyses.
PMCID: PMC107237  PMID: 9573170
9.  Identification of a Hotdog Fold Thioesterase Involved in the Biosynthesis of Menaquinone in Escherichia coli 
Journal of Bacteriology  2013;195(12):2768-2775.
Escherichia coli is used as a model organism for elucidation of menaquinone biosynthesis, for which a hydrolytic step from 1,4-dihydroxy-2-naphthoyl-coenzyme A (DHNA-CoA) to 1,4-dihydroxy-2-naphthoate is still unaccounted for. Recently, a hotdog fold thioesterase has been shown to catalyze this conversion in phylloquinone biosynthesis, suggesting that its closest homolog, YbgC in Escherichia coli, may be the DHNA-CoA thioesterase in menaquinone biosynthesis. However, this possibility is excluded by the involvement of YbgC in the Tol-Pal system and its complete lack of hydrolytic activity toward DHNA-CoA. To identify the hydrolytic enzyme, we have performed an activity-based screen of all nine Escherichia coli hotdog fold thioesterases and found that YdiI possesses a high level of hydrolytic activity toward DHNA-CoA, with high substrate specificity, and that another thioesterase, EntH, from siderophore biosynthesis exhibits a moderate, much lower DHNA-CoA thioesterase activity. Deletion of the ydiI gene from the bacterial genome results in a significant decrease in menaquinone production, which is little affected in ΔybgC and ΔentH mutants. These results support the notion that YdiI is the DHNA-CoA thioesterase involved in the biosynthesis of menaquinone in the model bacterium.
PMCID: PMC3697248  PMID: 23564174
10.  Crystallization and preliminary X-ray analysis of 6-­hydroxymethyl-7,8-dihydropterin pyrophosphokinase from Staphylococcus aureus  
The expression, purification and crystallization of S. aureus 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase, an essential enzyme from the folate-biosynthesis pathway, is reported.
6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the Mg2+-dependent transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-­dihydropterin (HMDP), forming 6-hydroxymethyl-7,8-dihydropterin pyro­phosphate, which is a critical step in the de novo folic acid-biosynthesis pathway. Diffraction-quality crystals of HPPK from the medically relevant species Staphylococcus aureus were grown in the presence of ammonium sulfate or sodium malonate and diffracted to better than 1.65 Å resolution. The crystals belonged to space group P21, with unit-cell parameters a = 36.8, b = 76.6, c = 51.5 Å, α = γ = 90.0, β = 100.2°. The crystals contained two molecules per asymmetric unit, with a volume per protein weight (V M) of 2.04 Å3 Da−1 and an estimated solvent content of 39.6%.
PMCID: PMC2864696  PMID: 20445263
HPPK; folate pathway; Staphylococcus aureus
11.  Comparative Genomics Guided Discovery of Two Missing Archaeal Enzyme Families Involved in the Biosynthesis of the Pterin Moiety of Tetrahydromethanopterin and Tetrahydrofolate 
ACS Chemical Biology  2012;7(11):1807-1816.
C-1 carriers are essential cofactors in all domains of life, and in Archaea, these can be derivatives of tetrahydromethanopterin (H4-MPT) or tetrahydrofolate (H4-folate). Their synthesis requires 6-hydroxymethyl-7,8-dihydropterin diphosphate (6-HMDP) as the precursor, but the nature of pathways that lead to its formation were unknown until the recent discovery of the GTP cyclohydrolase IB/MptA family that catalyzes the first step, the conversion of GTP to dihydroneopterin 2′,3′-cyclic phosphate or 7,8-dihydroneopterin triphosphate [El Yacoubi, B.; et al. (2006) J. Biol. Chem., 281, 37586–37593 and Grochowski, L. L.; et al. (2007) Biochemistry46, 6658–6667]. Using a combination of comparative genomics analyses, heterologous complementation tests, and in vitro assays, we show that the archaeal protein families COG2098 and COG1634 specify two of the missing 6-HMDP synthesis enzymes. Members of the COG2098 family catalyze the formation of 6-hydroxymethyl-7,8-dihydropterin from 7,8-dihydroneopterin, while members of the COG1634 family catalyze the formation of 6-HMDP from 6-hydroxymethyl-7,8-dihydropterin. The discovery of these missing genes solves a long-standing mystery and provides novel examples of convergent evolutions where proteins of dissimilar architectures perform the same biochemical function.
PMCID: PMC3500442  PMID: 22931285
12.  Comparative Genomics Guided Discovery of Two Missing Archaeal Enzyme Families Involved in the Biosynthesis of the Pterin Moiety of Tetrahydromethanopterin and Tetrahydrofolate 
ACS Chemical Biology  2012;7(11):1807-1816.
C-1 carriers are essential cofactors in all domains of life, and in Archaea, these can be derivatives of tetrahydromethanopterin (H4-MPT) or tetrahydrofolate (H4-folate). Their synthesis requires 6-hydroxymethyl-7,8-dihydropterin diphosphate (6-HMDP) as the precursor, but the nature of pathways that lead to its formation were unknown until the recent discovery of the GTP cyclohydrolase IB/MptA family that catalyzes the first step, the conversion of GTP to dihydroneopterin 2′,3′-cyclic phosphate or 7,8-dihydroneopterin triphosphate [El Yacoubi, B.; et al. (2006) J. Biol. Chem., 281, 37586–37593 and Grochowski, L. L.; et al. (2007) Biochemistry46, 6658–6667]. Using a combination of comparative genomics analyses, heterologous complementation tests, and in vitro assays, we show that the archaeal protein families COG2098 and COG1634 specify two of the missing 6-HMDP synthesis enzymes. Members of the COG2098 family catalyze the formation of 6-hydroxymethyl-7,8-dihydropterin from 7,8-dihydroneopterin, while members of the COG1634 family catalyze the formation of 6-HMDP from 6-hydroxymethyl-7,8-dihydropterin. The discovery of these missing genes solves a long-standing mystery and provides novel examples of convergent evolutions where proteins of dissimilar architectures perform the same biochemical function.
PMCID: PMC3500442  PMID: 22931285
13.  Structure of S. aureus HPPK and the Discovery of a New Substrate Site Inhibitor 
PLoS ONE  2012;7(1):e29444.
The first structural and biophysical data on the folate biosynthesis pathway enzyme and drug target, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (SaHPPK), from the pathogen Staphylococcus aureus is presented. HPPK is the second essential enzyme in the pathway catalysing the pyrophosphoryl transfer from cofactor (ATP) to the substrate (6-hydroxymethyl-7,8-dihydropterin, HMDP). In-silico screening identified 8-mercaptoguanine which was shown to bind with an equilibrium dissociation constant, Kd, of ∼13 µM as measured by isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). An IC50 of ∼41 µM was determined by means of a luminescent kinase assay. In contrast to the biological substrate, the inhibitor has no requirement for magnesium or the ATP cofactor for competitive binding to the substrate site. The 1.65 Å resolution crystal structure of the inhibited complex showed that it binds in the pterin site and shares many of the key intermolecular interactions of the substrate. Chemical shift and 15N heteronuclear NMR measurements reveal that the fast motion of the pterin-binding loop (L2) is partially dampened in the SaHPPK/HMDP/α,β-methylene adenosine 5′-triphosphate (AMPCPP) ternary complex, but the ATP loop (L3) remains mobile on the µs-ms timescale. In contrast, for the SaHPPK/8-mercaptoguanine/AMPCPP ternary complex, the loop L2 becomes rigid on the fast timescale and the L3 loop also becomes more ordered – an observation that correlates with the large entropic penalty associated with inhibitor binding as revealed by ITC. NMR data, including 15N-1H residual dipolar coupling measurements, indicate that the sulfur atom in the inhibitor is important for stabilizing and restricting important motions of the L2 and L3 catalytic loops in the inhibited ternary complex. This work describes a comprehensive analysis of a new HPPK inhibitor, and may provide a foundation for the development of novel antimicrobials targeting the folate biosynthetic pathway.
PMCID: PMC3261883  PMID: 22276115
14.  Bioengineering of Bacterial Polymer Inclusions Catalyzing the Synthesis of N-Acetylneuraminic Acid 
N-Acetylneuraminic acid is produced by alkaline epimerization of N-acetylglucosamine to N-acetylmannosamine and then subsequent condensation with pyruvate catalyzed by free N-acetylneuraminic acid aldolase. The high-alkaline conditions of this process result in the degradation of reactants and products, while the purification of free enzymes to be used for the synthesis reaction is a costly process. The use of N-acetylglucosamine 2-epimerase has been seen as an alternative to the alkaline epimerization process. In this study, these two enzymes involved in N-acetylneuraminic acid production were immobilized to biopolyester beads in vivo in a one-step, cost-efficient process of production and isolation. Beads with epimerase-only, aldolase-only, and combined epimerase/aldolase activity were recombinantly produced in Escherichia coli. The enzymatic activities were 32 U, 590 U, and 2.2 U/420 U per gram dry bead weight, respectively. Individual beads could convert 18% and 77% of initial GlcNAc and ManNAc, respectively, at high substrate concentrations and near-neutral pH, demonstrating the application of this biobead technology to fine-chemical synthesis. Beads establishing the entire N-acetylneuraminic acid synthesis pathway were able to convert up to 22% of the initial N-acetylglucosamine after a 50-h reaction time into N-acetylneuraminic acid.
PMCID: PMC3623141  PMID: 23455347
15.  Enhancement of 1,4-Dihydroxy-2-Naphthoic Acid Production by Propionibacterium freudenreichii ET-3 Fed-Batch Culture▿  
Applied and Environmental Microbiology  2007;73(10):3137-3143.
The production of 1,4-dihydroxy-2-naphthoic acid (DHNA) was investigated using a fed-batch culture of Propionibacterium freudenreichii ET-3. DHNA is a precursor of menaquinone (MK) and is transformed to MK by combination with an isoprenoid unit. We found that ET-3 stopped MK production and increased DHNA production in an anaerobic fed-batch culture by maintaining the lactose concentration at approximately zero. The maximum DHNA concentration observed in the anaerobic fed-batch culture was markedly higher than the maximum DHNA concentration observed in an anaerobic batch culture. Moreover, MK or DHNA production was affected by the lactose feeding rate; this suggests that lactose metabolism participates in the syntheses of these products. On the other hand, accumulation of propionate was found to inhibit DHNA production in the fed-batch culture. Based on the fact that ET-3 increases DHNA production in an aerobic culture by consuming propionate, we carried out a cultivation experiment in which an anaerobic fed-batch culture was switched to an anaerobic batch culture and found that the DHNA production was increased to a greater extent than the DHNA production in an anaerobic fed-batch culture. These results suggest that DHNA production by ET-3 is markedly influenced by carbon source limitation and the oxygen supply.
PMCID: PMC1907098  PMID: 17369348
16.  Exploring the Chemical Space around 8-Mercaptoguanine as a Route to New Inhibitors of the Folate Biosynthesis Enzyme HPPK 
PLoS ONE  2013;8(4):e59535.
As the second essential enzyme of the folate biosynthetic pathway, the potential antimicrobial target, HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase), catalyzes the Mg2+-dependant transfer of pyrophosphate from the cofactor (ATP) to the substrate, 6-hydroxymethyl-7,8-dihydropterin. Recently, we showed that 8-mercaptoguanine (8-MG) bound at the substrate site (KD ∼13 µM), inhibited the S. aureus enzyme (SaHPPK) (IC50 ∼ 41 µM), and determined the structure of the SaHPPK/8-MG complex. Here we present the synthesis of a series of guanine derivatives, together with their HPPK binding affinities, as determined by SPR and ITC analysis. The binding mode of the most potent was investigated using 2D NMR spectroscopy and X-ray crystallography. The results indicate, firstly, that the SH group of 8-MG makes a significant contribution to the free energy of binding. Secondly, direct N9 substitution, or tautomerization arising from N7 substitution in some cases, leads to a dramatic reduction in affinity due to loss of a critical N9-H···Val46 hydrogen bond, combined with the limited space available around the N9 position. The water-filled pocket under the N7 position is significantly more tolerant of substitution, with a hydroxyl ethyl 8-MG derivative attached to N7 (compound 21a) exhibiting an affinity for the apo enzyme comparable to the parent compound (KD ∼ 12 µM). In contrast to 8-MG, however, 21a displays competitive binding with the ATP cofactor, as judged by NMR and SPR analysis. The 1.85 Å X-ray structure of the SaHPPK/21a complex confirms that extension from the N7 position towards the Mg2+-binding site, which affords the only tractable route out from the pterin-binding pocket. Promising strategies for the creation of more potent binders might therefore include the introduction of groups capable of interacting with the Mg2+ centres or Mg2+ -binding residues, as well as the development of bitopic inhibitors featuring 8-MG linked to a moiety targeting the ATP cofactor binding site.
PMCID: PMC3614987  PMID: 23565155
17.  Bisubstrate analogue inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: New design with improved properties 
6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), a key enzyme in the folate biosynthetic pathway, catalyzes the pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin. The enzyme is essential for microorganisms, is absent from humans, and is not the target for any existing antibiotics. Therefore, HPPK is an attractive target for developing novel antimicrobial agents. Previously, we characterized the reaction trajectory of HPPK-catalyzed pyrophosphoryl transfer and synthesized a series of bisubstrate analog inhibitors of the enzyme by linking 6-hydroxymethylpterin to adenosine through 2, 3, or 4 phosphate groups. Here, we report a new generation of bisubstrate analog inhibitors. To improve protein binding and linker properties of such inhibitors, we have replaced the pterin moiety with 7,7-dimethyl-7,8-dihydropterin and the phosphate bridge with a piperidine linked thioether. We have synthesized the new inhibitors, measured their Kd and IC50 values, determined their crystal structures in complex with HPPK, and established their structure-activity relationship. 6-Carboxylic acid ethyl ester-7,7-dimethyl-7,8-dihydropterin, a novel intermediate that we developed recently for easy derivatization at position 6 of 7,7-dimethyl-7,8-dihydropterin, offers a much high yield for the synthesis of bisubstrate analogs than that of previously established procedure.
PMCID: PMC3257516  PMID: 22169600
Antibacterial; Bisubstrate; Folate; HPPK; Pterin
18.  Direct Observation of an Enamine Intermediate in Amine Catalysis 
Journal of the American Chemical Society  2009;131(51):18206-18207.
An enamine intermediate is believed to be the central feature of biological catalysts, such as aldolases and small molecule amine organocatalysts. Despite decades of investigation of naturally occurring aldolase enzymes and recent studies on designed aldolase antibodies and organocatalysts, direct structural observation of an enamine intermediate has proven to be rare. Herein, we report observation of a stable enamine intermediate in the crystal structure of an aldolase antibody 33F12 in complex with a 1,3-diketone derivative. This enamine complex structure provides strong evidence that fewer residues are essential for amine catalysis within the hydrophobic environments of this catalytic antibody than speculated for natural aldolase enzymes, and should serve to guide future studies aimed at the rational design of these types of catalysts, as well as organocatalysts. Indeed, enamine catalysis in proteins might be more simplistic than previously imagined.
PMCID: PMC3227542  PMID: 19968282
19.  Plasmodium falciparum: a paradigm for alternative folate biosynthesis in diverse microorganisms? 
Trends in parasitology  2008;24(11):502-508.
Folates have a key role in metabolism, and the folate-dependent generation of DNA precursors in the form of deoxythymidine 5′-phosphate is particularly important for the replication of malaria parasites. Although Plasmodium falciparum can synthesize folate derivatives de novo, a long-standing mystery has been the apparent absence of a key enzyme, dihydroneopterin aldolase, in the classical folate biosynthetic pathway of this organism. The discovery that a different enzyme, pyruvoyltetrahydropterin synthase, can produce the necessary substrate for the subsequent step in folate synthesis raises the question of whether this solution is unique to P. falciparum. Bioinformatic analyses suggest otherwise and indicate that an alternative route to folate could be widespread among diverse microorganisms and could be a target for novel drugs.
PMCID: PMC2720532  PMID: 18805734
20.  Detection of 1,4-Dihydroxy-2-Naphthoic Acid from Commercial Makgeolli Products 
To support beneficial effects of makgeolli for human health, we investigated for the presence of 1,4-dihydroxy-2-naphthoic acid (DHNA), a bifidogenic growth stimulator (BGS), from commercial makgeolli products. Among eleven makgeolli products (A∼K), four showed positive peaks for DHNA in high performance liquid chromatography analysis. Makgeolli product A in particular contained the highest concentration of DHNA (0.44 ppm), as confirmed by liquid chromatography-mass spectrometry. Furthermore, BGS activity of the makgeolli product A was higher than those of products in which DHNA was not detected. These results indicate that makgeolli can be a good source for DHNA and that DHNA-enriched makgeolli could be developed by modifying manufacturing procedures and controlling its microbiota.
PMCID: PMC3866769  PMID: 24471067
makgeolli; 1,4-dihydroxy-2-naphthoic acid; bifidogenic growth stimulator
21.  Sulfonamide Resistance in Streptococcus pyogenes Is Associated with Differences in the Amino Acid Sequence of Its Chromosomal Dihydropteroate Synthase 
Sulfonamide resistance in recent isolates of Streptococcus pyogenes was found to be associated with alterations of the chromosomally encoded dihydropteroate synthase (DHPS). There were 111 different nucleotides (13.8%) in the genes found in susceptible and resistant isolates, respectively, resulting in 30 amino acid changes (11.3%). These substantial changes suggested the possibility of a foreign origin of the resistance gene, in parallel to what has already been found for sulfonamide resistance in Neisseria meningitidis. The gene encoding DHPS was linked to at least three other genes encoding enzymes of the folate pathway. These genes were in the order GTP cyclohydrolase, dihydropteroate synthase, dihydroneopterin aldolase, and hydroxymethyldihydropterin pyrophosphokinase. The nucleotide differences in genes from resistant and susceptible strains extended from the beginning of the GTP cyclohydrolase gene to the end of the gene encoding DHPS, an additional indication for gene transfer in the development of resistance. Kinetic measurements established different affinities for sulfathiazole for DHPS enzymes isolated from resistant and susceptible strains.
PMCID: PMC105745  PMID: 9593127
22.  Genetic Bypass of Aspergillus nidulans crzA Function in Calcium Homeostasis 
G3: Genes|Genomes|Genetics  2013;3(7):1129-1141.
After dephosphorylation by the phosphatase calcineurin, the fungal transcription factor CrzA enters the nucleus and activates the transcription of genes responsible for calcium homeostasis and many other calcium-regulated activities. A lack of CrzA confers calcium-sensitivity to the filamentous fungus Aspergillus nidulans. To further understand calcium signaling in filamentous fungi and to identify genes that interact genetically with CrzA, we selected for mutations that were able to suppress crzAΔ calcium intolerance and identified three genes. Through genetic mapping, gene sequencing, and mutant rescue, we were able to identify these as cnaB (encoding the calcineurin regulatory subunit), folA (encoding an enzyme involved in folic acid biosynthesis, dihydroneopterin aldolase), and scrC (suppression of crzA-, encoding a hypothetical protein). By using a calcium indicator, Fluo-3, we were able to determine that the wild-type and the suppressor strains were either able to regulate intracellular calcium levels or were able to take up and or store calcium correctly. The increased expression of calcium transporters, pmcA and/or pmcB, in suppressor mutants possibly enabled tolerance to high levels of calcium. Our results suggest that a cnaB suppressor mutation confers calcium tolerance to crzAΔ strains through restoration of calcium homeostasis. These results stress that in A. nidulans there are calcineurin-dependent and CrzA-independent pathways. In addition, it is possible that CrzA is able to contribute to the modulation of folic acid biosynthesis.
PMCID: PMC3704241  PMID: 23665873
Aspergillus nidulan; extragenic suppression; calcineurin; CrzA; folate biosynthesis
23.  Structure of fructose bisphosphate aldolase from Encephalitozoon cuniculi  
The eukaryotic parasite E. cuniculi expresses a fructose bisphosphate aldolase that crystallizes readily in the presence of the partial substrate analog phosphate. This aldolase–phosphate structure and that of the sugar-bound Schiff base are reported. E. cuniculi aldolase displays a dimeric structure rather than the expected tetrameric quaternary structure.
Fructose bisphosphate aldolose (FBPA) enzymes have been found in a broad range of eukaryotic and prokaryotic organisms. FBPA catalyses the cleavage of fructose 1,6-bisphosphate into glyceraldehyde 3-phosphate and dihydroxy­acetone phosphate. The SSGCID has reported several FBPA structures from pathogenic sources. Bioinformatic analysis of the genome of the eukaryotic microsporidian parasite Encephalitozoon cuniculi revealed an FBPA homolog. The structures of this enzyme in the presence of the native substrate FBP and also with the partial substrate analog phosphate are reported. The purified enzyme crystallized in 90 mM Bis-Tris propane pH 6.5, 18% PEG 3350, 18 mM NaKHPO4, 10 mM urea for the phosphate-bound form and 100 mM Bis-Tris propane pH 6.5, 20% PEG 3350, 20 mM fructose 1,6-­bisphosphate for the FBP-bound form. In both cases protein was present at 25 mg ml−1 and the sitting-drop vapour-diffusion method was used. For the FBP-bound form, a data set to 2.37 Å resolution was collected from a single crystal at 100 K. The crystal belonged to the orthorhombic space group C2221, with unit-cell parameters a = 121.46, b = 135.82, c = 61.54 Å. The structure was refined to a final free R factor of 20.8%. For the phosphate-bound form, a data set was collected to 2.00 Å resolution. The space group was also C2221 and the unit-cell parameters were a = 121.96, b = 137.61, c = 62.23 Å. The structure shares the typical barrel tertiary structure reported for previous FBPA structures and exhibits the same Schiff base in the active site. The quaternary structure is dimeric. This work provides a direct experimental result for the substrate-binding conformation of the product state of E. cuniculi FBPA.
PMCID: PMC3169402  PMID: 21904050
SSGCID; aldolases; substrate binding; fructose 1,6-bisphosphate; fructose bisphosphate aldolose
24.  Functional Promiscuity of the COG0720 Family 
ACS Chemical Biology  2011;7(1):197-209.
The biosynthesis of GTP derived metabolites such as tetrahydrofolate (THF), biopterin (BH4), and the modified tRNA nucleosides queuosine (Q) and archaeosine (G+) relies on several enzymes of the Tunnel-fold superfamily. A subset of these proteins include the 6-pyruvoyl-tetrahydropterin (PTPS-II), PTPS-III, and PTPS-I homologs, all members of the COG0720 family, that have been previously shown to transform 7,8-dihydroneopterin triphosphate (H2NTP) into different products. PTPS-II catalyzes the formation of 6-pyruvoyltetrahydropterin in the BH4 pathway. PTPS-III catalyzes the formation of 6-hydroxylmethyl-7,8-dihydropterin in the THF pathway. PTPS-I catalyzes the formation of 6-carboxy-5,6,7,8-tetrahydropterin in the Q pathway. Genes of these three enzyme families are often misannotated as they are difficult to differentiate by sequence similarity alone. Using a combination of physical clustering, signature motif, and phylogenetic co-distribution analyses, in vivo complementation studies, and in vitro enzymatic assays, a complete reannotation of the COG0720 family was performed in prokaryotes. Notably, this work identified and experimentally validated dual function PTPS-I/III enzymes involved in both THF and Q biosynthesis. Both in vivo and in vitro analyses showed that the PTPS-I family could tolerate a translation of the active site cysteine and was inherently promiscuous, catalyzing different reactions on the same substrate, or the same reaction on different substrates. Finally, the analysis and experimental validation of several archaeal COG0720 members confirmed the role of PTPS-I in archaeosine biosynthesis, and resulted in the identification PTPS-III enzymes with variant signature sequences in Sulfolobus species. This study reveals an expanded versatility of the COG0720 family members and illustrates that for certain protein families, extensive comparative genomic analysis beyond homology is required to correctly predict function.
PMCID: PMC3262898  PMID: 21999246
Queuosine; archaeosine; tetrahydrofolate; biopterin; tRNA modification; riboflavin; 6-pyruvoyl-tetrahydropterin synthase
25.  Propionibacterium freudenreichii component 1.4‐dihydroxy‐2‐naphthoic acid (DHNA) attenuates dextran sodium sulphate induced colitis by modulation of bacterial flora and lymphocyte homing 
Gut  2006;55(5):681-688.
Background and aim
1.4‐Dihydroxy‐2‐naphthoic acid (DHNA), a bifidogenic growth stimulator from Propionibacterium freudenreichii, is thought to have a beneficial effect as a prebiotic; however, its in vivo effect on intestinal inflammation remains unknown. The aim of this study was to determine whether oral administration of DHNA can ameliorate dextran sodium sulphate (DSS) induced colitis and to determine the possible underlying mechanisms.
Colitis was induced in mice by treatment with 2.0% DSS for seven days. DHNA (0.6 or 2.0 mg/kg) was given in drinking water prior to (preventive study) or after (therapeutic study) DSS administration. Colonic damage was histologically scored, and mucosal addressin cell adhesion molecule 1 (MAdCAM‐1) expression and β7 positive cell infiltration were determined by immunohistochemistry. mRNA levels of proinflammatory cytokines (interleukin (IL)‐1β, IL‐6 and tumour necrosis factor α (TNF‐α)) were determined by quantitative real time polymerase chain reaction. In addition, bacterial flora in the caecum, concentrations of short chain acids, and luminal pH were examined.
DHNA improved survival rate and histological damage score in mice administered DSS in both the preventive and therapeutic studies. DHNA significantly attenuated the enhanced expression of MAdCAM‐1, the increased β7 positive cell number, and the increased mRNA levels of IL‐1β, IL‐6, and TNF‐α in DSS treated colon. In addition, the decreased number of Lactobacillus and Enterobacteriaceae induced by DSS was recovered by DHNA. Preventive effects on decrease in butyrate concentration and decrease in pH level in mice administered DSS were also observed in the DHNA preventive study.
DHNA, a novel type of prebiotic, attenuates colonic inflammation not only by balancing intestinal bacterial flora but also by suppressing lymphocyte infiltration through reduction of MAdCAM‐1.
PMCID: PMC1856113  PMID: 16299037
mucosal addressin cell adhesion molecule 1; β7 integrin; prebiotics; bacterial flora; short chain fatty acids

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