Functional characterization of a methionine gamma-lyase in Arabidopsis and its implication in an alternative to the reverse trans-sulfuration pathway

Methionine gamma-lyase (MGL) catalyzes the degradation of L-methionine to alpha-ketobutyrate, methanethiol and ammonia. The Arabidopsis (Arabidopsis thaliana) genome includes a single gene (At1g64660) encoding a protein (AtMGL) with approximately 35% identity to bacterial and protozoan MGLs. When overexpressed in Escherichia coli, AtMGL allowed growth on L-methionine as sole nitrogen source and conferred a high rate of methanethiol emission. The purified recombinant protein exhibited a spectrum typical of pyridoxal 5'-phosphate enzymes, and had high activity toward l-methionine, L-ethionine, L-homocysteine and seleno-L-methionine, but not L-cysteine. Quantitation of mRNA showed that the AtMGL gene is expressed in aerial organs and roots, and that its expression in leaves was increased 2.5-fold by growth on low sulfate medium. Emission of methanethiol from Arabidopsis plants supplied with 10 mM L-methionine was undetectable (<0.5 nmol min(-1) g(-1) FW), suggesting that AtMGL is not an important source of volatile methanethiol. Knocking out the AtMGL gene significantly increased leaf methionine content (9.2-fold) and leaf and root S-methylmethionine content (4.7- and 7-fold, respectively) under conditions of sulfate starvation, indicating that AtMGL carries a significant flux in vivo. In Arabidopsis plantlets fed L-[(35)S]methionine on a low sulfate medium, label was incorporated into protein-bound cysteine as well as methionine, but incorporation into cysteine was significantly (30%) less in the knockout mutant. These data indicate that plants possess an alternative to the reverse trans-sulfuration pathway (methionine-->homocysteine-->cystathionine-->cysteine) in which methanethiol is an intermediate.     

Early lateral transfer of genes encoding malic enzyme, acetyl-CoA synthetase and alcohol dehydrogenases from anaerobic prokaryotes to Entamoeba histolytica

The fermentation enzymes, which enable the microaerophilic protist Entamoeba histolytica to parasitize the colonic lumen and tissue abscesses, closely resemble homologues in anaerobic prokaryotes. Here, genes encoding malic enzyme and acetyl-CoA synthetase (nucleoside diphosphate forming) were cloned from E. histolytica, and their evolutionary origins, as well as those encoding two alcohol dehydrogenases (ADHE and ADH1), were inferred by means of phylogenetic reconstruction. The E. histolytica malic enzyme, which decarboxylates malate to pyruvate, closely resembles that of the archaeon Archaeoglobus fulgidus, strongly suggesting a common origin. The E. histolytica acetyl-CoA synthetase, which converts acetyl-CoA to acetate with the production of ATP, appeared to be closely related to the Plasmodium falciparum enzyme, but it was no more closely related to the Giardia lamblia acetyl-CoA synthetase than to those of archaea. Phylogenetic analyses suggested that the adh1 and adhe genes of E. histolytica and Gram-positive eubacteria share a common ancestor. Lateral transfer of genes encoding these fermentation enzymes from archaea or eubacteria to E. histolytica probably occurred early, because the sequences of the amoebic enzymes show considerable divergence from those of prokaryotes, and the amoebic genes encoding these enzymes are in the AT-rich codon usage of the parasite.     

Characterization of the gene encoding serine acetyltransferase, a regulated enzyme of cysteine biosynthesis from the protist parasites Entamoeba histolytica and Entamoeba dispar. Regulation and possible function of the cysteine biosynthetic pathway in Entamoeba.

The enteric protist parasites Entamoeba histolytica and Entamoeba dispar possess a cysteine biosynthetic pathway, unlike their mammalian host, and are capable of de novo production of L-cysteine. We cloned and characterized cDNAs that encode the regulated enzyme serine acetyltransferase (SAT) in this pathway from these amoebae by genetic complementation of a cysteine-auxotrophic Escherichia coli strain with the amoebic cDNA libraries. The deduced amino acid sequences of the amoebic SATs exhibited, within the most conserved region, 36-52% identities with the bacterial and plant SATs. The amoebic SATs contain a unique insertion of eight amino acids, also found in the corresponding region of a plasmid-encoded SAT from Synechococcus sp., which showed the highest overall identities to the amoebic SATs. Phylogenetic reconstruction also revealed a close kinship of the amoebic SATs with cyanobacterial SATs. Biochemical characterization of the recombinant E. histolytica SAT revealed several enzymatic features that distinguished the amoebic enzyme from the bacterial and plant enzymes: 1) inhibition by L-cysteine in a competitive manner with L-serine; 2) inhibition by L-cystine; and 3) no association with cysteine synthase. Genetically engineered amoeba strains that overproduced cysteine synthase and SAT were created. The cysteine synthase-overproducing amoebae had a higher level of cysteine synthase activity and total thiol content and revealed increased resistance to hydrogen peroxide. These results indicate that the cysteine biosynthetic pathway plays an important role in antioxidative defense of these enteric parasites.     

Partial purification and some properties of a neutral sulfhydryl and an acid proteinase from Entamoeba histolytica.

The partial purification of two intracellular proteinases from the protozoan parasite Entamoeba histolytica is reported. One of these enzymes is an acid proteinase exhibiting maximum activity at pH 3.5 (hemoglobin substrate), is little affected by a range of inhibitors or activators, and is presumed to be similar to cathepsin D. Also present is a neutral proteinase exhibiting optimum activity at pH 6.0 (azocasein) but only poorly hydrolyzing either hemoglobin or serum albumen. This latter enzyme displayed no metal ion requirement, but was markedly inhibited by thiol-blocking agents and activated by free sulhydryl-containing compounds.     

Molecular and biochemical characterization of D-phosphoglycerate dehydrogenase from Entamoeba histolytica. A unique enteric protozoan parasite that possesses both phosphorylated and nonphosphorylated serine metabolic pathways.

A putative phosphoglycerate dehydrogenase (PGDH), which catalyzes the oxidation of d-phosphoglycerate to 3-phosphohydroxypyruvate in the so-called phosphorylated serine metabolic pathway, from the enteric protozoan parasite Entamoeba histolytica was characterized. The E. histolytica PGDH gene (EhPGDH) encodes a protein of 299 amino acids with a calculated molecular mass of 33.5 kDa and an isoelectric point of 8.11. EhPGDH showed high homology to PGDH from bacteroides and another enteric protozoan ciliate, Entodinium caudatum. EhPGDH lacks both the carboxyl-terminal serine binding domain and the 13-14 amino acid regions containing the conserved Trp139 (of Escherichia coli PGDH) in the nucleotide binding domain shown to be crucial for tetramerization, which are present in other organisms including higher eukaryotes. EhPGDH catalyzed reduction of phosphohydroxypyruvate to phosphoglycerate utilizing NADH and, less efficiently, NADPH; EhPGDH did not utilize 2-oxoglutarate. Kinetic parameters of EhPGDH were similar to those of mammalian PGDH, for example the preference of NADH cofactor, substrate specificities and salt-reversible substrate inhibition. In contrast to PGDH from bacteria, plants and mammals, the EhPGDH protein is present as a homodimer as demonstrated by gel filtration chromatography. The E. histolytica lysate contained PGDH activity of 26 nmol NADH utilized per min per mg of lysate protein in the reverse direction, which consisted 0.2-0.4% of a total soluble protein. Altogether, this parasite represents a unique unicellular protist that possesses both phosphorylated and nonphosphorylated serine metabolic pathways, reinforcing the biological importance of serine metabolism in this organism. Amino acid sequence comparison and phylogenetic analysis of various PGDH sequences showed that E. histolytica forms a highly supported monophyletic group with another enteric protozoa, cilliate E. caudatum, and bacteroides.     

Purification and characterization of phenylacetate-coenzyme A ligase from a denitrifying Pseudomonas sp., an enzyme involved in the anaerobic degradation of phenylacetate

The enzyme catalysing the first step in the anaerobic degradation pathway of phenylacetate was purified from a denitrifying Pseudomonas strain KB 740. It catalyses the reaction phenylacetate+CoA+ATP phenylacetyl-CoA+AMP+PP_i and requires Mg²⁺. Phenylacetate-CoA ligase (AMP forming) was found in cells grown anaerobically with phenylacetate and nitrate. Maximal specific enzyme activity was 0.048 mol min^-1 x mg^-1 protein in the mid-exponential growth phase. After 640-fold purification with 18% yield, a specific activity of 24.4 mol min^-1 mg^-1 protein was achieved. The enzyme is a single polypeptide with Mr of 52 ±2 kDa. The purified enzyme shows high specificity towards the aromatic inducer substrate phenylacetate and uses ATP preferentially; Mn²⁺ can substitute for Mg²⁺. The apparent K _m values for phenylacetate, CoA, and ATP are 60, 150, and 290 M, respectively. The soluble enzyme has an optimum pH of 8.5, is insensitive to oxygen, but is rather labile and requires the presence of glycerol and/or phenylacetate for stabilization. The N-terminal amino acid sequence showed no homology to other reported CoA-ligases. The expression of the enzye was studied by immunodetection. It is present in cells grown anaerobically with phenylacetate, but not with mandelate, phenylglyoxylate, benzoate; small amounts were detected in cells grown aerobically with phenylacetate.     

Purification and characterization of mouse alcohol dehydrogenase from two inbred strains that differ in total liver enzyme activity

Alcohol dehydrogenase activity in mouse liver homogenate-supernatants is 1.7 times greater in the C57BL/10 strain than in the BALB/c strain, regardless of whether activity is expressed in units per gram liver, total liver, or milligram DNA. The K _m values for ethanol and NAD⁺, approximately 0.4 and 0.03mm, respectively, of enzyme purified from both strains are similar. Moreover, the K _i for NADH, 1 µm, the pH optimum for ethanol oxidation, 10.5, and the V _max for ethanol oxidation, 160 min^–1, for ADH from the C57BL/10 and BALB/c strains are similar. Therefore, the difference in ADH activity in the two strains cannot be due to differences in the catalytic properties of the enzyme. The electrophoretic and isoelectric focusing patterns and two-dimensional tryptic peptide maps of the purified enzyme from both strains are identical. Thus the amino acid sequences of enzyme from C57BL/10 and BALB/c mice must also be identical or very similar. The difference in ADH activity in the two strains is most likely the result of genetic differences in the content of ADH protein in liver.Supported by NIAAA Grant AA 04307.     

Identification and characterization of two families of F420H2‐dependent reductases from Mycobacteria that catalyse aflatoxin degradation

Aflatoxins are polyaromatic mycotoxins that contaminate a range of food crops as a result of fungal growth and contribute to serious health problems in the developing world because of their toxicity and mutagenicity. Although relatively resistant to biotic degradation, aflatoxins can be metabolized by certain species of Actinomycetales. However, the enzymatic basis for their breakdown has not been reported until now. We have identified nine Mycobacterium smegmatis enzymes that utilize the deazaflavin cofactor F₄₂₀H₂ to catalyse the reduction of the α,β‐unsaturated ester moiety of aflatoxins, activating the molecules for spontaneous hydrolysis and detoxification. These enzymes belong to two previously uncharacterized F₄₂₀H₂ dependent reductase (FDR‐A and ‐B) families that are distantly related to the flavin mononucleotide (FMN) dependent pyridoxamine 5′‐phosphate oxidases (PNPOxs). We have solved crystal structures of an enzyme from each FDR family and show that they, like the PNPOxs, adopt a split barrel protein fold, although the FDRs also possess an extended and highly charged F₄₂₀H₂ binding groove. A general role for these enzymes in xenobiotic metabolism is discussed, including the observation that the nitro‐reductase Rv3547 from Mycobacterium tuberculosis that is responsible for the activation of bicyclic nitroimidazole prodrugs belongs to the FDR‐A family.     

Cloning and functional characterization of an enzyme from Helicobacter pylori that catalyzes two steps of the methylerythritol phosphate pathway for isoprenoid biosynthesis

Background

The methylerythritol phosphate pathway for isoprenoid biosynthesis is an attractive target for the design of new specific antibiotics for the treatment of gastrointestinal diseases associated with the presence of the bacterium Helicobacter pylori since this pathway which is essential to the bacterium is absent in humans.

Results

This work reports the molecular cloning of one of the genes of the methylerythritol phosphate pathway form H. pylori (ispDF; HP_1440) its expression in Escherichia coli and the functional characterization of the recombinant enzyme. As shown by genetic complementation and in vitro functional assays the product of the ispDF gene form H. pylori is a bifunctional enzyme which can replace both CDP-methylerythritol synthase and methylerythritol cyclodiphosphate synthase from E. coli.

General significance

Designing inhibitors that affect at the same time both enzyme activities of the H. pylori bifunctional enzyme (i.e. by disrupting protein oligomerization) would result in more effective antibiotics which would be able to continue their action even if the bacterium acquired a resistance to another antibiotic directed against one of the individual activities.

Conclusion

The bifunctional enzyme would be an excellent target for the design of new, selective antibiotics for the treatment of H. pylori associated diseases.     

Identification and characterization of an aromatic amino acid decarboxylase from the filarial nematode, Dirofilaria immitis

A novel secreted aromatic amino acid decarboxylase-like molecule was identified in the excretory/secretory products of L3/L4 larvae as well as in an extract of adult Dirofilaria immitis. The secretion of the enzyme was developmentally regulated. Peak enzyme activities were detected in the culture medium before and after the molting of L3 larvae in vitro. The enzyme was purified from D. immitis adult extracts and the excretory/secretory products of L3/L4 larvae using different chromatographic methods followed by isoelectric focusing and SDS-PAGE. The enzyme has a molecular mass of 48 kDa and a pI of 5.6, and shows a specific enzymatic activity towards the aromatic amino acid substrates phenylalanine, tyrosine and tryptophan. The enzyme's activity did not show an absolute requirement for exogenous pyridoxal-5-phosphate. However, addition of pyridoxal-5-phosphate at 5 microM in the reaction increased the enzyme activity greatly. The enzyme had the ability to catalyze the formation of dopamine from L-dopa. Studies on the effects of inhibitors on the enzyme activity showed that the enzyme was sensitive to Pefabloc and p-chloromercuribenzoic acid, but not to diisopropyl flurophosphate. The Km values of the enzyme for H-Phe-AMC, H-Tyr-AMC and H-Trp-AMC were calculated to be 32.1 microM, 35.1 microM and 29.1 microM, respectively.     

Identification and biochemical characterization of two novel UDP-2,3-diacetamido-2,3-dideoxy-alpha-D-glucuronic acid 2-epimerases from respiratory pathogens

For a long period lactate was considered as a dead-end product of glycolysis in many cells and its accumulation correlated with acidosis and cellular and tissue damage. At present, the role of lactate in several physiological processes has been investigated based on its properties as an energy source, a signalling molecule and as essential for tissue repair. It is noteworthy that lactate accumulation alters glycolytic flux independently from medium acidification, thereby this compound can regulate glucose metabolism within cells. PFK (6-phosphofructo-1-kinase) is the key regulatory glycolytic enzyme which is regulated by diverse molecules and signals. PFK activity is directly correlated with cellular glucose consumption. The present study shows the property of lactate to down-regulate PFK activity in a specific manner which is not dependent on acidification of the medium. Lactate reduces the affinity of the enzyme for its substrates, ATP and fructose 6-phosphate, as well as reducing the affinity for ATP at its allosteric inhibitory site at the enzyme. Moreover, we demonstrated that lactate inhibits PFK favouring the dissociation of enzyme active tetramers into less active dimers. This effect can be prevented by tetramer-stabilizing conditions such as the presence of fructose 2,6-bisphosphate, the binding of PFK to f-actin and phosphorylation of the enzyme by protein kinase A. In conclusion, our results support evidence that lactate regulates the glycolytic flux through modulating PFK due to its effects on the enzyme quaternary structure.     

Identification of a key amino acid in hemagglutinin that increases human-type receptor binding and transmission of an H6N2 avian influenza virus

Binding exclusively to human-type receptors is a prerequisite for avian influenza viruses to transmit from human to human. We previously reported that 34% of H6 avian influenza viruses recognize the human-type receptor, but their affinity for the avian-type receptor remains higher than that for the human-type receptor. Here, we found that a single amino acid change from glutamine to leucine at position 226 of hemagglutinin caused a switch in receptor-binding preference from avian-type to human-type receptors and rendered A/chicken/Guangdong/S1312/2010(H6N2) capable of respiratory droplet transmission in guinea pigs.     

Identification and characterization of the human cytomegalovirus immediate-early region 2 gene that stimulates gene expression from an inducible promoter.

The human cytomegalovirus (HCMV) XbaI E cloned DNA fragment of approximately 20 kilobases can complement an adenovirus mutant (dl312) defective in the E1a viral gene product (D. J. Spector and M. J. Tevethia, Virology 151:329-338, 1986). This viral DNA fragment contains three immediate-early (IE) genes between 0.709 and 0.751 map units (M. F. Stinski, D. R. Thomsen, R. M. Stenberg, and L. C. Goldstein, J. Virol. 46:1-14, 1983). Two of the IE genes, IE1 and IE2, were isolated and tested for a role in regulating viral gene expression. Since HCMV early and late promoters require additional characterization, the chloramphenicol acetyl transferase (cat) gene, driven by the adenovirus E2 promoter, was used as an indicator of gene expression. cat expression from this heterologous viral promoter was shown to be stimulated by HCMV at early times after infection. The IE1 gene product did not function independently in activating this promoter. The IE2 gene products could independently stimulate the expression of a plasmid of a plasmid when the cat gene was placed downstream of the inducible E2 promoter (E2CAT). Five proteins of different sizes have been predicted to originate from IE2, depending on mRNA splicing. The protein products specified by the IE2 gene were characterized with an antibody to a synthetic peptide according to the open reading frame of exon 2. Three of the five proteins are encoded by exon 2. Three viral proteins of 82, 54, and 28 kilodaltons (kDa) were detected. The exons contained in the region designated as IE2a have open reading frames that could code for two of the smaller proteins of 27 and 30 kDa. This region, when driven by the HCMV enhancer, could independently stimulate gene expression from E2CAT to a high level. A plasmid with the HCMV enhancer upstream of exons, that could code for the HCMV IE2 proteins of 48 and 51 kDa, as well as 27- and 30-kDa proteins, also stimulated E2CAT expression but at a lower level. The activity of this plasmid was augmented by the IE1 gene product, despite the fact that the latter gene product alone was inactive. It is proposed that the HCMV IE region 2 gene products are involved in the regulation of viral or host cell promoters either independently or in combination with other HCMV IE proteins.     

Biochemical and biophysical characterization of 1,4-naphthoquinone as a dual inhibitor of two key enzymes of type II fatty acid biosynthesis from Moraxella catarrhalis

The fatty acid biosynthesis (FAS II) is a vital process in bacteria and regarded as an attractive pathway for the development of potential antimicrobial agents. In this study, we report 1,4-naphthoquinone (NPQ) as a dual inhibitor of two key enzymes of FAS II pathway, namely FabD (Malonyl-CoA:ACP transacylase) and FabZ (β-hydroxyacyl-ACP dehydratase). Mode of inhibition of NPQ was found to be non-competitive for both enzymes with IC₅₀ of 26.67 μΜ and 23.18 μΜ against McFabZ and McFabD respectively. Conformational changes in secondary and tertiary structures marked by the loss of helical contents were observed in both enzymes upon NPQ binding. The fluorescence quenching was found to be static with a stable ground state complex formation. ITC based studies have shown that NPQ is binding to McFabZ with a stronger affinity (~1.5×) as compared to McFabD. Molecular docking studies have found that NPQ interacts with key residues of both McFabD (Ser209, Arg126, and Leu102) and McFabZ (His74 and Tyr112) enzymes. Both complexes have shown the structural stability during the 20 ns run of molecular dynamics based simulations. Altogether, the present study suggests that NPQ scaffold can be exploited as a multi-targeted inhibitor of FAS II pathway, and these biochemical and biophysical findings will further help in the development of potent antibacterial agents targeting FAS II pathway.     

Taxol biosynthesis: Identification and characterization of two acetyl CoA:taxoid-O-acetyl transferases that divert pathway flux away from Taxol production

Two cDNAs encoding taxoid-O-acetyl transferases (TAX 9 and TAX 14) were obtained from a previously isolated family of Taxus acyl/aroyl transferase cDNA clones. The recombinant enzymes catalyze the acetylation of taxadien-5α,13α-diacetoxy-9α,10β-diol to generate taxadien-5α,10β,13α-tri-acetoxy-9α-ol and taxadien-5α,9α,13α-triacetoxy-10β-ol, respectively, both of which then serve as substrates for a final acetylation step to yield taxusin, a prominent side-route metabolite of Taxus. Neither enzyme acetylate the 5α- or the 13α-hydroxyls of taxoid polyols, indicating that prior acylations is required for efficient peracetylation to taxusin. Both enzymes were kinetically characterized, and the regioselectivity of acetylation was shown to vary with pH. Sequence comparison with other taxoid acyl transferases confirmed that primary structure of this enzyme type reveals little about function in taxoid metabolism. Unlike previously identified acetyl transferases involved in Taxol production, these two enzymes appear to act exclusively on partially acetylated taxoid polyols to divert the Taxol pathway to side-route metabolites.     

Identification of conserved histidines and glutamic acid as key residues for isomerohydrolase activity of RPE65, an enzyme of the visual cycle in the retinal pigment epithelium

We have recently reported that RPE65 from the retinal pigment epithelium is the isomerohydrolase, a critical enzyme in the visual cycle for regeneration of 11-cis retinal, the chromophore for visual pigments. Here, we demonstrated that mutation of any one of the absolutely conserved four histidine and one glutamic acid residues to alanine in RPE65 abolished its isomerohydrolase activity. Substitution of the conserved glutamic acid with glutamine also resulted in loss of the activity. Moreover, these mutations significantly reduced protein stability of RPE65. These results indicate that these conserved residues are essential for the isomerohydrolase activity of RPE65 and its stability.