Dihydroneopterin aldolase (DHNA) catalyzes the conversion of 7,8-dihydroneopterin to 6-hydroxymethyl-7,8-dihydropterin and also the epimerization of DHNP to 7,8-dihydromonapterin. Previously, we determined the crystal structure of Staphylococcus aureus DHNA (SaDHNA) in complex with the substrate analogue neopterin (NP). We also showed that Escherichia coli DHNA (EcDHNA) and SaDHNA have significantly different binding and catalytic properties by biochemical analysis. On the basis of these structural and functional data, we proposed a catalytic mechanism involving two proton wires.
To understand the structural basis for the biochemical differences and further investigate the catalytic mechanism of DHNA, we have determined the structure of EcDHNA complexed with NP at 1.07-Å resolution [PDB:2O90], built an atomic model of EcDHNA complexed with the substrate DHNP, and performed molecular dynamics (MD) simulation analysis of the substrate complex. EcDHNA has the same fold as SaDHNA and also forms an octamer that consists of two tetramers, but the packing of one tetramer with the other is significantly different between the two enzymes. Furthermore, the structures reveal significant differences in the vicinity of the active site, particularly in the loop that connects strands β3 and β4, mainly due to the substitution of nearby residues. The building of an atomic model of the complex of EcDHNA and the substrate DHNP and the MD simulation of the complex show that some of the hydrogen bonds between the substrate and the enzyme are persistent, whereas others are transient. The substrate binding model and MD simulation provide the molecular basis for the biochemical behaviors of the enzyme, including noncooperative substrate binding, indiscrimination of a pair of epimers as the substrates, proton wire switching during catalysis, and formation of epimerization product.
The EcDHNA and SaDHNA structures, each in complex with NP, reveal the basis for the biochemical differences between EcDHNA and SaDHNA. The atomic substrate binding model and MD simulation offer insights into substrate binding and catalysis by DHNA. The EcDHNA structure also affords an opportunity to develop antimicrobials specific for Gram-negative bacteria, as DHNAs from Gram-negative bacteria are highly homologous and E. coli is a representative of this class of bacteria.
Dihydroneopterin aldolase; DHNA; Structure; Dynamics; Catalysis
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.
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.
Folate biosynthesis; Aldehyde-Lyases; 7,8-dihydroneopterin; Tuberculosis; Kinetics; Solvent Kinetic Isotope Effects; Nuclear Magnetic Resonance; Oxygen chemistry; Oxygenases
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.
The crystal structures of two 1,4-dihydroxy-2-naphthoyl-CoA thioesterases of plant and cyanobacterial origin that are involved in the biosynthesis of phylloquinone are presented. The divergent structures of these two functionally similar enzymes indicate convergent evolution.
The synthesis of phylloquinone (vitamin K1) in photosynthetic organisms requires a thioesterase that hydrolyzes 1,4-dihydroxy-2-naphthoyl-CoA (DHNA-CoA) to release 1,4-dihydroxy-2-naphthoate (DHNA). Cyanobacteria and plants contain distantly related hotdog-fold thioesterases that catalyze this reaction, although the structural basis of these convergent enzymatic activities is unknown. To investigate this, the crystal structures of hotdog-fold DHNA-CoA thioesterases from the cyanobacterium Synechocystis (Slr0204) and the flowering plant Arabidopsis thaliana (AtDHNAT1) were determined. These enzymes form distinct homotetramers and use different active sites to catalyze hydrolysis of DHNA-CoA, similar to the 4-hydroxybenzoyl-CoA (4-HBA-CoA) thioesterases from Pseudomonas and Arthrobacter. Like the 4-HBA-CoA thioesterases, the DHNA-CoA thioesterases contain either an active-site aspartate (Slr0204) or glutamate (AtDHNAT1) that are predicted to be catalytically important. Computational modeling of the substrate-bound forms of both enzymes indicates the residues that are likely to be involved in substrate binding and catalysis. Both enzymes are selective for DHNA-CoA as a substrate, but this selectivity is achieved using divergent predicted binding strategies. The Slr0204 binding pocket is predominantly hydrophobic and closely conforms to DHNA, while that of AtDHNAT1 is more polar and solvent-exposed. Considered in light of the related 4-HBA-CoA thioesterases, these structures indicate that hotdog-fold thioesterases using either an active-site aspartate or glutamate diverged into distinct clades prior to the evolution of strong substrate specificity in these enzymes.
thioesterases; hotdog fold; vitamin K; phylloquinone; Synechocystis; Arabidopsis thaliana
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.
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.
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.
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.
mucosal addressin cell adhesion molecule 1; β7 integrin; prebiotics; bacterial flora; short chain fatty acids
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.
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.
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.
Substrate specificity of enzymes is frequently narrow and constrained by multiple interactions, limiting the use of natural enzymes in biocatalytic applications. Aldolases have important synthetic applications, but the usefulness of these enzymes is hampered by their narrow reactivity profile with unnatural substrates. To explore the determinants of substrate selectivity and alter the specificity of E. coli 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, we employed structure-based mutagenesis coupled with library screening of mutant enzymes localized to the bacterial periplasm. We identified two active site mutations (T161S/S184L) that work additively to enhance the substrate specificity of this aldolase to include catalysis of retro-aldol cleavage of (4S)-2-keto-4-hydroxy-4-(2′-pyridyl)butyrate (S-KHPB). These mutations improve the value of kcat/KMS-KHPB by >450-fold, resulting in a catalytic efficiency that is comparable to that of the wild-type enzyme with the natural substrate while retaining high stereoselectivity. Moreover, the value of kcatS-KHPB for this mutant enzyme, a parameter critical for biocatalytic applications, is 3-fold higher than the maximum value achieved by the natural aldolase with any substrate. This mutant also possesses high catalytic efficiency for the retro-aldol cleavage of the natural substrate, KDPG, and a >50-fold improved activity for cleavage of 2-keto-4-hydroxy-octonoate (KHO), a non-functionalized hydrophobic analog. These data suggest a substrate binding mode that illuminates the origin of facial selectivity in aldol addition reactions catalyzed by KDPG and 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolases. Furthermore, targeting mutations to the active site provides marked improvement in substrate selectivity, demonstrating that structure-guided active site mutagenesis combined with selection techniques can efficiently identify proteins with characteristics that compare favorably to naturally occurring enzymes.
rational redesign; directed evolution; protein engineering; biocatalysis; stereoselectivity
2-Keto-3-deoxy-6-phosphogluconate (KDPG) and 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolases catalyze an identical reaction differing in substrate specificity in only the configuration of a single stereocenter. However, the proteins show little sequence homology at the amino acid level. Here we investigate the determinants of substrate selectivity of these enzymes. The Escherichia coli KDPGal aldolase gene, cloned into a T7 expression vector and overexpressed in E. coli, catalyzes retro-aldol cleavage of the natural substrate, KDPGal, with values of kcat/KM and kcat of 1.9 × 104 M−1 s−1 and 4 s−1, respectively. In the synthetic direction, KDPGal aldolase efficiently catalyzes an aldol addition using a limited number of aldehyde substrates, including D-glyceraldehyde-3-phosphate (natural substrate), D-glyceraldehyde, glycolaldehyde, and 2-pyridinecarboxaldehyde. A preparative scale reaction between 2-pyridinecarboxaldehyde and pyruvate catalyzed by KDPGal aldolase produced the aldol adduct of the R stereochemistry in >99.7% ee, a result complementary to that observed using the related KDPG aldolase. The native crystal structure has been solved to a resolution of 2.4Å and displays the same (α/β)8 topology, as KDPG aldolase. We have also determined a 2.1Å structure of a Schiff base complex between the enzyme and its substrate. This model predicts that a single amino acid change, T161 in KDPG aldolase to V154 in KDPGal aldolase, plays an important role in determining the stereochemical course of enzyme catalysis and this prediction was borne out by site-directed mutagenesis studies. However, additional changes in the enzyme sequence are required to prepare an enzyme with both high catalytic efficiency and altered stereochemistry.
KDPGal aldolase; (α/β)8 topology; Aldol addition; Stereoselectivity
2-Keto-3-deoxy-6-phosphogluconate (KDPG) and 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolases catalyze an identical reaction differing in substrate specificity in only the configuration of a single stereocenter. However, the proteins show little sequence homology at the amino acid level. Here we investigate the determinants of substrate selectivity of these enzymes. The Escherichia coli KDPGal aldolase gene, cloned into a T7 expression vector and overexpressed in E. coli, catalyzes retro-aldol cleavage of the natural substrate, KDPGal, with values of kcat/KM and kcat of 1.9 × 104 M−1 s−1 and 4 s−1, respectively. In the synthetic direction, KDPGal aldolase efficiently catalyzes an aldol addition using a limited number of aldehyde substrates, including D-glyceraldehyde-3-phosphate (natural substrate), D-glyceraldehyde, glycolaldehyde, and 2-pyridinecarboxaldehyde. A preparative scale reaction between 2-pyridinecarboxaldehyde and pyruvate catalyzed by KDPGal aldolase produced the aldol adduct of the Rstereochemistry in >99.7% ee, a result complementary to that observed using the related KDPG aldolase. The native crystal structure has been solved to a resolution of 2.4 Å and displays the same (α/β)8 topology, as KDPG aldolase. We have also determined a 2.1 Å structure of a Schiff base complex between the enzyme and its substrate. This model predicts that a single amino acid change, T161 in KDPG aldolase to V154 in KDPGal aldolase, plays an important role in determining the stereochemical course of enzyme catalysis and this prediction was borne out by site-directed mutagenesis studies. However, additional changes in the enzyme sequence are required to prepare an enzyme with both high catalytic efficiency and altered stereochemistry.
C-1 carriers are essential cofactors in all domains of
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.
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.
The industrially important polysaccharide alginate is composed of the two sugar monomers β-d-mannuronic acid (M) and its epimer α-l-guluronic acid (G). In the bacterium Azotobacter vinelandii, the G residues originate from a polymer-level reaction catalyzed by one periplasmic and at least five secreted mannuronan C-5-epimerases. The secreted enzymes are composed of repeats of two protein modules designated A (385 amino acids) and R (153 amino acids). The modular structure of one of the epimerases, AlgE1, is A1R1R2R3A2R4. This enzyme has two catalytic sites for epimerization, each site introducing a different G distribution pattern, and in this article we report the DNA-level construction of a variety of truncated forms of the enzyme. Analyses of the properties of the corresponding proteins showed that an A module alone is sufficient for epimerization and that A1 catalyzed the formation of contiguous stretches of G residues in the polymer, while A2 introduces single G residues. These differences are predicted to strongly affect the physical and immunological properties of the reaction product. The epimerization reaction is Ca2+ dependent, and direct binding studies showed that both the A and R modules bind this cation. The R modules appeared to reduce the Ca2+ concentration needed for full activity and also stimulated the reaction rate when positioned both N and C terminally.
Hereditary fructose intolerance (HFI) is a metabolic disorder caused by enzymic deficiency of aldolase B, a genetically distinct cytosolic isoenzyme expressed exclusively in liver, kidney, and intestine. The molecular basis of this enzyme defect has been investigated in three affected individuals from a nonconsanguineous kindred, in whom fructose-l-phosphate aldolase activities in liver or intestinal biopsy samples were reduced to 2-6% of mean control values.
To identify a putative enzyme mutant in tissue extracts, aldolase B was purified from human liver by affinity chromatography and monospecific antibodies were prepared from antiserum raised in sheep. Immunodiffusion gels showed a single precipitin line common to pure enzyme and extracts of normal liver and intestine, but no reaction with extracts of brain, muscle, or HFI liver. However, weak positive staining for aldolase in hepatocyte and enterocyte cytosol was demonstrated by indirect immunofluorescence of HFI tissues. This was abolished by pretreatment with pure enzyme protein. Accordingly, a specific radioimmunoassay (detection limit 7.5 ng) was established to quantify immunoreactive aldolase B in human biopsy specimens. Extracts of tissue from affected patients gave 10-25% immunoreactive enzyme in control samples; immunoreactive aldolase in intestinal extracts from four heterozygotes was reduced (to 55%) when compared with seven samples from normal control subjects (P < 0.05). In extracts of HFI tissues, there was a sevenfold reduction in apparent absolute specific activity (1.02 vs. 8.82 U/mg) of immunoreactive fructose-l-phosphate aldolase B, but the apparent specific activity in heterozygotes (7.71 U/mg) was only slightly impaired. Displacement radioimmunotitration of aldolase B in liver supernatants showed a significant (P < 0.005) decrease in antibody avidity for immunoreactive protein in HFI tissue when compared with the pure enzyme or extract of normal control liver.
Immunoaffinity chromatography on antialdolase B-Sepharose facilitated isolation and purification of enzyme from liver biopsy specimens. Active aldolase in normal liver, with substrate activity ratios and Michaelis constants identical to biochemically purified human enzyme, could be recovered from antibody columns. Chromatography on monospecific Fab' antialdolase B enabled pure enzyme protein to be retrieved quantitatively from normal control and HFI liver: direct chemical assay showed 1.88 and 1.15 mg aldolase protein/g of tissue, respectively. This confirmed that the catalytic properties of the HFI aldolase were profoundly impaired with specific activities of fructose-l-phosphate cleavage of 7.21 and 0.07 U/mg, respectively. Radioimmunoassay gave estimates of 7.66 and 1.18 U/mg, respectively. Sodium dodecyl sulfate-polyacrylamide electrophoresis indicated that immunopurified aldolase from HFI liver possessed a single subunit size similar to material from control liver extracts: Mr 39,100 vs. 37,900±700 (SD) D, respectively. Electrofocusing under denaturing conditions of aldolase isolated in parallel from control and HFI liver revealed the same complement of subunits and, despite qualitative differences in distribution of bands during degradation, no additional charged species.
Fructose phosphate aldolase deficiency in hereditary fructose intolerance is attended by the synthesis of an immunoreactive, but functionally and structurally modified enzyme variant that results from a restricted genetic mutation.
Three catabolic enzymes, UlaD, UlaE, and UlaF, are involved in a pathway leading to fermentation of l-ascorbate under anaerobic conditions. UlaD catalyzes a β-keto acid decarboxylation reaction to produce l-xylulose-5-phosphate, which undergoes successive epimerization reactions with UlaE (l-xylulose-5-phosphate 3-epimerase) and UlaF (l-ribulose-5-phosphate 4-epimerase), yielding d-xylulose-5-phosphate, an intermediate in the pentose phosphate pathway. We describe here crystallographic studies of UlaE from Escherichia coli O157:H7 that complete the structural characterization of this pathway. UlaE has a triosephosphate isomerase (TIM) barrel fold and forms dimers. The active site is located at the C-terminal ends of the parallel β-strands. The enzyme binds Zn2+, which is coordinated by Glu155, Asp185, His211, and Glu251. We identified a phosphate-binding site formed by residues from the β1/α1 loop and α3′ helix in the N-terminal region. This site differs from the well-characterized phosphate-binding motif found in several TIM barrel superfamilies that is located at strands β7 and β8. The intrinsic flexibility of the active site region is reflected by two different conformations of loops forming part of the substrate-binding site. Based on computational docking of the l-xylulose 5-phosphate substrate to UlaE and structural similarities of the active site of this enzyme to the active sites of other epimerases, a metal-dependent epimerization mechanism for UlaE is proposed, and Glu155 and Glu251 are implicated as catalytic residues. Mutation and activity measurements for structurally equivalent residues in related epimerases supported this mechanistic proposal.
Carbohydrate epimerization is an essential technology for the widespread production of rare sugars. In contrast to other enzymes, most epimerases are only active on sugars substituted with phosphate or nucleotide groups, thus drastically restricting their use. Here we show that Sn-Beta zeolite in the presence of sodium tetraborate catalyses the selective epimerization of aldoses in aqueous media. Specifically, a 5 wt% aldose (for example, glucose, xylose or arabinose) solution with a 4:1 aldose:sodium tetraborate molar ratio reacted with catalytic amounts of Sn-Beta yields near-equilibrium epimerization product distributions. The reaction proceeds by way of a 1,2 carbon shift wherein the bond between C-2 and C-3 is cleaved and a new bond between C-1 and C-3 is formed, with C-1 moving to the C-2 position with an inverted configuration. This work provides a general method of performing carbohydrate epimerizations that surmounts the main disadvantages of current enzymatic and inorganic processes.
Epimerization of carbohydrates to rare sugars yields products that have potential applications as anti-viral drugs or chiral building blocks. Here, Sn-Beta zeolite in the presence of sodium tetraborate is shown to catalyze the selective epimerization of aldoses in aqueous media.
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.
Queuosine; archaeosine; tetrahydrofolate; biopterin; tRNA modification; riboflavin; 6-pyruvoyl-tetrahydropterin synthase
Giardia lamblia fructose-1,6-bisphosphate aldolase (FBPA)1 is a member of the Class II zinc-dependent aldolase family that catalyzes the cleavage of D-fructose-1,6-bisphosphate (FBP) into dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (G3P). In addition to the active site zinc, the catalytic apparatus of FBPA employs an aspartic acid, Asp83 in the G. lamblia enzyme, which when replaced by an alanine residue renders the enzyme inactive. A comparison of the crystal structures of the D83A FBPA in complex with FBP and of the wild-type FBPA in the unbound state revealed a substrate induced conformational transition of loops in the vicinity of the active site and a shift in the location of Zn2+. Upon FBP binding, the Zn2+ shifts up to 4.6 Å towards the catalytic Asp83, which brings the metal within coordination distance to the Asp83 carboxylate group. In addition, the structure of wild-type FBPA was determined in complex with the competitive inhibitor D-tagatose 1,6-bisphosphate (TBP), a FBP stereoisomer. In this structure, the zinc binds in a site close to that previously seen in the structure of FBPA in complex with phosphoglycolohydroxamate, an analog of the postulated DHAP ene-diolate intermediate. Together, the ensemble of structures suggests that the zinc mobility is necessary to orient the Asp83 side chain and to polarize the substrate for proton transfer from the FBP C(4) hydroxyl group to the Asp83 carboxyl group. In the absence of FBP, the alternative zinc position is too remote for coordinating the Asp83. We propose a modification of the catalytic mechanism that incorporates the novel features observed in the FBPA/FBP structure. The mechanism invokes coordination and co-planarity of the Zn2+ with the FBP’s O-C(3)-C(4)-O concomitant with coordination of Asp83 carboxylic group. Catalysis is accompanied by movement of Zn2+ to a site co-planar with the O-C(2)-C(3)-O of the DHAP. glFBPA exhibit strict substrate specificity towards FBP and does not cleave TBP. The active sites of FBPAs contain an aspartate residue equivalent to Asp255 of glFBPA, whereas tagatose-1,6-bisphosphate aldolase contains an alanine in this position. We and others hypothesized that this aspartic acid is a likely determinant of FBP vs. TBP specificity. Replacement of Asp255 by an alanine resulted in an enzyme that possesses double specificity, now cleaving TBP (albeit with low efficacy; kcat/Km = 80 M−1s−1) while maintaining activity towards FBP at 50-fold lower catalytic efficacy compared with the wild type FBPA. The collection of structures and sequence analyses highlighted additional residues that may be involved in substrate discrimination.
The polysaccharide alginate forms a protective capsule for Pseudomonas aeruginosa during chronic pulmonary infections. The structure of alginate, a linear polymer of β1-4-linked O-acetylated d-mannuronate (M) and l-guluronate (G), is important for its activity as a virulence factor. Alginate structure is mediated by AlgG, a periplasmic C-5 mannuronan epimerase. AlgG also plays a role in protecting alginate from degradation by the periplasmic alginate lyase AlgL. Here, we show that the C-terminal region of AlgG contains a right-handed β-helix (RHβH) fold, characteristic of proteins with the carbohydrate-binding and sugar hydrolase (CASH) domain. When modeled based on pectate lyase C of Erwinia chrysanthemi, the RHβH of AlgG has a long shallow groove that may accommodate alginate, similar to protein/polysaccharide interactions of other CASH domain proteins. The shallow groove contains a 324-DPHD motif that is conserved among AlgG and the extracellular mannuronan epimerases of Azotobacter vinelandii. Point mutations in this motif disrupt mannuronan epimerase activity but have no effect on alginate secretion. The D324A mutation has a dominant negative phenotype, suggesting that the shallow groove in AlgG contains the catalytic face for epimerization. Other conserved motifs of the epimerases, 361-NNRSYEN and 381-NLVAYN, are predicted to lie on the opposite side of the RHβH from the catalytic center. Point mutations N362A, N367A, and V383A result in proteins that do not protect alginate from AlgL, suggesting that these mutant proteins are not properly folded or not inserted into the alginate biosynthetic scaffold. These motifs are likely involved in asparagine and hydrophobic stacking, required for structural integrity of RHβH proteins, rather than for mannuronan catalysis. The results suggest that the AlgG RHβH protects alginate from degradation by AlgL by channeling the alginate polymer through the proposed alginate biosynthetic scaffold while epimerizing approximately every second d-mannuronate residue to l-guluronate along the epimerase catalytic face.
Catalytic Atom Maps (CAMs) are minimal models of enzyme active sites. The structures in the Protein Data Bank (PDB) were examined to determine if proteins with CAM-like geometries in their active sites all share the same catalytic function. We combined the CAM-based search protocol with a filter based on the weighted contact number (WCN) of the catalytic residues, a measure of the “crowdedness” of the microenvironment around a protein residue. Using this technique, a CAM based on the Ser-His-Asp catalytic triad of trypsin was able to correctly identify catalytic triads in other enzymes within 0.5 Å RMSD of the Catalytic Atom Map with 96% accuracy. A CAM based on the Cys-Arg-(Asp/Glu) active site residues from the tyrosine phosphatase active site achieved 89% accuracy in identifying this type of catalytic functionality. Both of these Catalytic Atom Maps were able to identify active sites across different fold types. Finally, the PDB was searched to locate proteins with catalytic functionality similar to that present in the active site of orotidine 5′-monophosphate decarboxylase (ODCase), whose mechanism is not known with certainty. A CAM, based on the conserved Lys-Asp-Lys-Asp tetrad in the ODCase active site, was used to search the PDB for enzymes with similar active sites. The ODCase active site has a geometry similar to that of Schiff base-forming Class I aldolases, with lowest aldolase RMSD to the ODCase CAM at 0.48 Å. The similarity between this CAM and the aldolase active site suggests that ODCase has the correct catalytic functionality present in its active site for the generation of a nucleophilic lysine.