Unique properties of Plasmodium falciparum porphobilinogen deaminase

The hybrid pathway for heme biosynthesis in the malarial parasite proposes the involvement of parasite genome-coded enzymes of the pathway localized in different compartments such as apicoplast, mitochondria, and cytosol. However, knowledge on the functionality and localization of many of these enzymes is not available. In this study, we demonstrate that porphobilinogen deaminase encoded by the Plasmodium falciparum genome (PfPBGD) has several unique biochemical properties. Studies carried out with PfPBGD partially purified from parasite membrane fraction, as well as recombinant PfPBGD lacking N-terminal 64 amino acids expressed and purified from Escherichia coli cells (DeltaPfPBGD), indicate that both the proteins are catalytically active. Surprisingly, PfPBGD catalyzes the conversion of porphobilinogen to uroporphyrinogen III (UROGEN III), indicating that it also possesses uroporphyrinogen III synthase (UROS) activity, catalyzing the next step. This obviates the necessity to have a separate gene for UROS that has not been so far annotated in the parasite genome. Interestingly, DeltaPfP-BGD gives rise to UROGEN III even after heat treatment, although UROS from other sources is known to be heat-sensitive. Based on the analysis of active site residues, a DeltaPfPBGDL116K mutant enzyme was created and the specific activity of this recombinant mutant enzyme is 5-fold higher than DeltaPfPBGD. More interestingly, DeltaPfPBGDL116K catalyzes the formation of uroporphyrinogen I (UROGEN I) in addition to UROGEN III, indicating that with increased PBGD activity the UROS activity of PBGD may perhaps become rate-limiting, thus leading to non-enzymatic cyclization of preuroporphyrinogen to UROGEN I. PfPBGD is localized to the apicoplast and is catalytically very inefficient compared with the host red cell enzyme.     

Maize uroporphyrinogen III methyltransferase: overexpression of the functional gene fragments in Escherichia coli and one-step purification

S-Adenosyl-L-methionine: uroporphyrinogen III methyltransferase (SUMT), a key regulatory enzyme, converts uroporphyrinogen III to precorrin-2 in the porphinoids biosynthesis. In this study, the mature SUMT was signified that the maize SUMT precursor encoded by the open reading frame of maize SUMT cDNA was deleted the first 91 amino acids constituting the postulated signal peptide. Several mature SUMT fusion and deletion mutants were conducted. It actively expressed in Escherichia coli that the mature SUMT, or the truncated one deleting the C-terminal extra 52 amino acids based on SUMT sequence comparisons. On the contrary, it expressed as an inclusion body in E. coli that the mature SUMT fusion mutant, the SUMT precursor, or the mature SUMT deleting the N-terminal 36 amino acids including glycine-rich region involved directly in SAM binding. The purified His6-tagged mature SUMT was homodimer with a molecular weight of 34 kDa, as shown by SDS-PAGE, 52 kDa using gel-filtration chromatography, and 79 kDa by dynamic light scattering assay. Red fluorescent compounds were associated with the recombinant mature SUMT which were identified as sirohydrochlorin and trimethylpyrrocorphin by spectroscopic analysis. This association slightly altered the protein secondary structure confirmed by circular dichroism assay.     

Identification,cloning, and sequencing of the genes involved in the conversion of D,L-2-amino-delta2-thiazoline-4-carboxylic acid to L-cysteine in Pseudomonas sp. strain ON-4a

The newly isolated strain Pseudomonas sp. ON-4a converts D,L-2-amino-delta2-thiazoline-4-carboxylic acid to L-cysteine via N-carbamoyl-L-cysteine. A genomic DNA fragment from this strain containing the gene(s) encoding enzymes that convert D,L-2-amino-delta2-thiazoline-4-carboxylic acid into L-cysteine was cloned in Escherichia coli. Transformants expressing cysteine-forming activity were selected by growth of an E. coli mutant defective in the cysB gene. A positive clone, denoted CM1, carrying the plasmid pCM1 with an insert DNA of approximately 3.4 kb was obtained, and the nucleotide sequence of a complementing region was analyzed. Analysis of the sequence found two open reading frames, ORF1 and ORF2, which encoded proteins of 183 and 435 amino acid residues, respectively. E. coli DH5alpha harboring pTrCM1, which was constructed by inserting the subcloned sequence into an expression vector, expressed two proteins of 25 kDa and 45 kDa. From the analyses of crude extracts of E. coli DH5alpha carrying deletion derivatives of pTrCM1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by enzymatic activity, it was found that the 25-kDa protein encoded by ORF1 was the enzyme L-2-amino-delta2-thiazoline-4-carboxylic acid hydrolase, which catalyzes the conversion of L-2-amino-delta2-thiazoline-4-carboxylic acid to N-carbamoyl-L-cysteine, and that the 45-kDa protein encoded by ORF2 was the enzyme N-carbamoyl-L-cysteine amidohydrolase, which catalyzes the conversion of N-carbamoyl-L-cysteine to L-cysteine.     

Analysis of a cDNA encoding arginine decarboxylase from oat reveals similarity to the Escherichia coli arginine decarboxylase and evidence of protein processing

Summary Arginine decarboxylase is the first enzyme in one of the two pathways of putrescine synthesis in plants. We purified arginine decarboxylase from oat leaves, obtained N-terminal amino acid sequence, and then used this information to isolate a cDNA encoding oat arginine decarboxylase. Comparison of the derived amino acid sequence with that of the arginine decarboxylase gene from Escherichia coli reveals several regions of sequence similarity which may play a role in enzyme function. The open reading frame (ORF) in the oat cDNA encodes a 66 kDa protein, but the arginine decarboxylase polypeptide that we purified has an apparent molecular weight of 24 kDa and is encoded in the carboxyl-terminal region of the ORF. A portion of the cDNA encoding this region was expressed in E. coli, and a polyclonal antibody was developed against the expressed polypeptide. The antibody detects 34 kDa and 24 kDa polypeptides on Western blots of oat leaf samples. Maturation of arginine decarboxylase in oats appears to include processing of a precursor protein.     

An FMN hydrolase of the haloacid dehalogenase superfamily is active in plant chloroplasts

FMN hydrolases catalyze dephosphorylation of FMN to riboflavin. Although these enzymes have been described in many organisms, few had their corresponding genes cloned and their recombinant proteins biochemically characterized, and none had their physiological roles determined. We found previously that FMN hydrolase activity in pea chloroplasts is Mg(2+)-dependent, suggesting an enzyme of the haloacid dehalogenase (HAD) superfamily. In this study, a new FMN hydrolase was purified by multistep chromatography after ammonium sulfate precipitation. The molecular weight of the native protein was estimated at ～59,400, a dimer of about twice the predicted molecular weight of most HAD superfamily phosphatases. After SDS-PAGE of the partially purified material, two separate protein bands within 25-30 kDa were extracted from the gel and analyzed by nanoLC-MS/MS. Peptide sequence matching to the protein samples suggested the presence of three HAD-like hydrolases. cDNAs for sequence homologs from Arabidopsis thaliana of these proteins were expressed in Escherichia coli. Activity screening of the encoded proteins showed that the At1g79790 gene encodes an FMN hydrolase (AtcpFHy1). Plastid localization of AtcpFHy1 was confirmed using fluorescence microscopy of A. thaliana protoplasts transiently expressing the N-terminal fusion of AtcpFHy1 to enhanced green fluorescent protein. Phosphatase activity of AtcpFHy1 is FMN-specific, as assayed with 19 potential substrates. Kinetic parameters and pH and temperature optima for AtcpFHy1 were determined. A phylogenetic analysis of putative phosphatases of the HAD superfamily suggested distinct evolutionary origins for the plastid AtcpFHy1 and the cytosolic FMN hydrolase characterized previously.     

Identification and biochemical characterization of plant acylamino acid-releasing enzyme

Plant acylamino acid-releasing enzyme (AARE) catalyzing the N-terminal hydrolysis of N(alpha)-acylpeptides to release N(alpha)-acylated amino acids, was biochemically characterized using recombinant and native AAREs. A cDNA encoding a deduced Arabidopsis thaliana AARE (AtAARE) was cloned and sequenced. The deduced amino acid sequence encoded a 764 amino acid protein of 83.9 kDa, which was 31.8% identical with that of rat AARE. In particular, the proposed catalytic residues (Ser, Asp, and His) of AARE, called the "catalytic triad residues, " were completely conserved. Recombinant AtAARE was expressed in Escherichia coli and confirmed to be a functional AARE. Native AAREs were prepared from A. thaliana and cucumber (Cucumis sativus, L.) plants. Both native AAREs were tetrameric proteins of 350 kDa comprising four subunits of 82 kDa, and showed typical enzymological properties of other AAREs, i.e. sensitivity to diisopropyl fluorophosphate, an optimum pH of around 7.0, and an optimum temperature of 37 degrees C. Both the native and recombinant AAREs were immunochemically homologous. Intracelluar fractionation analysis showed that the AARE was mainly present in the stroma of chloroplasts. Native AARE degraded the glycated ribulose-1,5-bisphoshate carboxylase/oxygenase protein but not the native protein. Thus, plant AARE might be involved in not only catalysis of the N-terminal hydrolysis of N(alpha)-acylpeptides but also the elimination of glycated proteins.     

SepA, the major extracellular protein of Shigella flexneri: autonomous secretion and involvement in tissue invasion

In addition to lpa proteins and lcsA, which are involved in entry into epithelial cells and intercellular spread, respectively, Shigella secretes a 110 kDa protein, designated SepA. We report the identification, cloning, and nucleotide sequence determination of the sepA gene, analysis of SepA secretion, and construction and characterization of a sepA mutant. The sepA gene is carried by the virulence plasmid and codes for a 150 kDa precursor. Upon secretion, which does not involve accessory proteins encoded by the virulence plasmid, the precursor is converted to a mature protein of 110 kDa by two cleavages removing an N-terminal signal sequence and a C-terminal fragment. Extensive similarities were detected between the sequence of the first 500 residues of mature SepA and the N-terminal region of lgA1 proteases from Neisseria gonorrhoeae and Haemophilus influenzae , the Tsh haemagglutinin of an avian pathogenic Escherichia coli , and the Hap protein involved in adhesion and penetration of H. influenzae . The C-terminal domain of the SepA precursor, which is not present in the secreted protein, exhibits sequence similarity with pertactin of Bordetella pertussis and the ring-forming protein of Helicobacter mustelae . Construction and phenotypic characterization of a sepA mutant indicated that SepA is required neither for entry into cultured epithelial cells nor for intercellular dissemination. However, in the rabbit ligated ileal loop model, the sepA mutant exhibited an attenuated virulence, which suggests that SepA might play a role in tissue invasion.     

NADH: ubiquinone oxidoreductase from bovine heart mitochondria. A fourth nuclear encoded subunit with a homologue encoded in chloroplast genomes.

The amino acid sequence has been determined of the precursor of a nuclear encoded 20 kDa subunit of complex I from bovine heart mitochondria. The sequence of the mature protein is related to a protein of uncertain function, hitherto known as psbG, encoded in the chloroplast genomes of higher plants. Open reading frames encoding homologues of psbG have also been detected in bacteria and in the mitochondrial genome of Paramecium tetraurelia. The chloroplast psbG gene is found between ndhC and ndhJ, which encode homologues of ND3, a hydrophobic subunit of complex I encoded in the bovine mitochondrial genome, and of the nuclear encoded 30 kDa subunit of complex I. This 20 kDa protein is the eleventh out of the forty or more subunits of bovine complex I with a chloroplast encoded homologue, and its sequence provides further support for the presence in chloroplasts of a multisubunit enzyme related to complex I that could be involved in chlororespiration. The strict conservation of three cysteines suggests that the subunit might be an iron-sulphur protein.     

Nucleoside diphosphate kinase from pea chloroplasts: purification,cDNA cloning and import into chloroplasts

Nucleoside diphosphate kinase (NDPK; EC 2.7.4.6) was enriched 1900-fold from purified pea (Pisum sativum L. cv. Golf.) chloroplasts. The active enzyme preparation contained two polypeptides of apparent molecular weight 18.5 kDa and 17.4kDa. Both proteins were enzymatically active and were recognized by an antiserum raised against NDPK from spinach chloroplasts, suggesting the existence of two isoforms in pea chloroplasts. The N-terminal protein sequence data were obtained for both polypeptides and compared with the nucleotide sequence of a cDNA clone isolated from a pea cDNA library. The analysis revealed that the two NDPK forms are encoded for by one mRNA, indicating that the lower-molecular-weight form could represent a proteolytic breakdown product of the 18.5-kDa NDPK. The pea chloroplastic NDPK is made as a larger precursor protein which is imported into chloroplasts. The NDPK precursor is then processed by the stromal processing peptidase to yield the 18.5-kDa form.Abbreviations NDPK nucleoside diphosphate kinase - preNDPK precursor NDPK - ps-NDPK cDNA coding for Pisum sativum NDPK II We thank Dr. Schmidt, University Göttingen, Germany, for doing the protein sequencing. This work was supported in part by grants from the Deutsche Forschungsgemeinschaft.     

The 100 kDa haem:haemopexin-binding protein of Haemophilus Influenzae: structure and localization

All Haemophilus influenzae strains have an absolute requirement for exogenously supplied haem for aerobic growth. A majority of strains of H. influenzae type b (Hib) produce a 100 kDa protein which binds haem: haemopexin complexes. This 100 kDa haem:haemopexin binding protein, designated HxuA, was originally detected on the Hib cell surface. Monoclonal antibody (mAb)-based analyses revealed that the HxuA protein was also present in soluble form in Hib culture supernatants. This soluble HxuA protein exhibited haem:haemopexin-binding activity in a direct binding assay. Nucleotide sequence analysis of the hxuA gene from Hib strain DL42, together with N-terminal amino acid analysis of HxuA protein purified from Hib culture supernatant, revealed that this protein was synthesized as a 101 kDa precursor with a leader peptide that was removed to yield a 99kDa protein. Southern blot analysis of chromosomal DNA from four Hib and four non-typeable H. influenzae (NTHI) strains detected the presence of a single band in each strain that hybridized a Hib hxuA gene probe. Subsequent analysis of these NTHI strains showed that all four strains released into culture supernatant a haem:haemopexin-binding protein that migrated in SDS-PAGE at a rate similar or identical to that of the Hib HxuA protein. A Hib hxuA mutant was used to screen an NTHI genomic DNA library and an NTHI gene was cloned that complemented the mutation in this Hib strain. Nucleotide sequence analysis of this NTHI gene revealed that it encoded a protein with 87% identity to the Hib HxuA protein. The expression of HxuA by both Hib and NTHI strains indicates that this particular haem acquisition system is conserved among H. influenzae strains.     

Identification and characterization of YLR328W, the Saccharomyces cerevisiae structural gene encoding NMN adenylyltransferase. Expression and characterization of the recombinant enzyme.

The enzyme nicotinamide mononucleotide (NMN) adenylyltransferase (EC 2.7.7.1) catalyzes the transfer of the adenylyl moiety of ATP to NMN to form NAD. A new purification procedure for NMN adenylyltransferase from Saccharomyces cerevisiae provided sufficient amounts of enzyme for tryptic fragmentation. Through data-base search a full matching was found between the sequence of tryptic fragments and the sequence of a hypothetical protein encoded by the S. cerevisiae YLR328W open reading frame (GenBank accession number U20618). The YLR328W gene was isolated, cloned into a T7-based vector and successfully expressed in Escherichia coli BL21 cells, yielding a high level of NMN adenylyltransferase activity. The purification of recombinant protein, by a two-step chromatographic procedure, resulted in a single polypeptide of 48 kDa under SDS-PAGE, in agreement with the molecular mass of the hypothetical protein encoded by YLR328W ORF. The N-terminal sequence of the purified recombinant NMN adenylyltransferase exactly corresponds to the predicted sequence. Molecular and kinetic properties of recombinant NMN adenylyltransferase are reported and compared with those already known for the enzyme obtained from different sources.     

Expression and analysis of the human cytomegalovirus UL80-encoded protease: identification of autoproteolytic sites.

The 45-kDa assembly protein of human cytomegalovirus is encoded by the C-terminal portion of the UL80 open reading frame (ORF). For herpes simplex virus, packaging of DNA is accompanied by cleavage of its assembly protein precursor at a site near its C terminus, by a protease encoded by the N-terminal region of the same ORF (F. Liu and B. Roizman, J. Virol. 65:5149-5156, 1991). By analogy with herpes simplex virus, we investigated whether a protease is contained within the N-terminal portion of the human cytomegalovirus UL80 ORF. The entire UL80 ORF was expressed in Escherichia coli, under the control of the phage T7 promoter. UL80 should encode a protein of 85 kDa. Instead, the wild-type construct produces a set of proteins with molecular masses of 50, 30, 16, 13, and 5 kDa. In contrast, when mutant UL80 is deleted of the first 14 amino acids, it produces only an 85-kDa protein. These results suggest that the UL80 polyprotein undergoes autoproteolysis. We demonstrate by deletional analysis and by N-terminal sequencing that the 30-kDa protein is the protease and that it originates from the N terminus of UL80. The UL80 polyprotein is cleaved at the following three sites: (i) at the C terminus of the assembly protein domain, (ii) between the 30- and 50-kDa proteins, and (iii) within the 30-kDa protease itself, which yields the 16- and 13-kDa proteins and may be a mechanism to inactivate the protease.     

Haemoglobin receptor protein is intragenically encoded by the cysteine proteinase-encoding genes and the haemagglutinin-encoding gene of Porphyromonas gingivalis

The obligately anaerobic bacterium Porphyromonas gingivalis produces characteristic black-pigmented colonies on blood agar. It is thought that the black pigmentation is caused by haem accumulation and is related to virulence of the microorganism. P. gingivalis cells expressed a prominent 19 kDa protein when grown on blood agar plates. Analysis of its N-terminal amino acid sequence indicated that the 19 kDa protein was encoded by an internal region (HGP15 domain) of an arginine-specific cysteine proteinase (Arg-gingipain, RGP)-encoding gene ( rgp1 ) and was also present in genes for lysine-specific cysteine proteinases ( prtP and kgp ) and a haemagglutinin ( hagA ) of P. gingivalis . The HGP15 domain protein was purified from an HGP15-overproducing Escherichia coli and was found to have the ability to bind to haemoglobin in a pH-dependent manner. The anti-HGP15 antiserum reacted with the 19 kDa haemoglobin-binding protein in the envelope of P. gingivalis. P. gingivalis wild-type strain showed pH-dependent haemoglobin adsorption, whereas its non-pigmented mutants that produced no HGP15-related proteins showed deficiency in haemoglobin adsorption. These results strongly indicate a close relationship among HGP15 production, haemoglobin adsorption and haem accumulation of P. gingivalis .     

Isolation,sequencing and expression of cDNA sequences encoding uroporphyrinogen decarboxylase from tobacco and barley

We have cloned and sequenced a full-length cDNA for uroporphyrinogen decarboxylase (UROD, EC 4.1.1.37) from tobacco (Nicotiana tabacum L.) and a partial cDNA clone from barley (Hordeum vulgare L.). The cDNA of tobacco encodes a protein of 43 kDa, which has 33% overall similarity to UROD sequences determined from other organisms. We propose that tobacco UROD has an N-terminal extension of 39 amino acid residues. This extension is most likely a chloroplast transit sequence. The in vitro translation product of UROD was imported into pea chloroplasts and processed to ca. 39 kDa. A truncated cDNA, from which the putative transit peptide had been deleted, was used to over-express the mature UROD in Escherichia coli. Purified protein showed UROD activity, thus providing an adequate source for subsequent enzymatic characterization and inhibition studies. Expression of UROD was investigated by northern and western blot analysis during greening of etiolated barley seedlings, and in segments of barley primary leaves grown under day/night cycles. The amount of RNA and protein increased during illumination Maximum UROD-RNA levels were detected in the basal segments relative to the top of the leaf.Abbreviations ALA 5-aminolevulinic acid - copro coproporphyrin - coprogen coproporphyrinogen - protogen IX protoporphyrinogen IX - UROD uroporphyrinogen decarboxylase - uro uroporphyrin - urogen uroporphyrinogen     

Molecular cloning of the gene encoding an outer-membrane-associated beta-N-acetylglucosaminidase involved in chitin degradation system of Alteromonas sp. strain O-7

The gene encoding beta-N-acetylglucosaminidase (GlcNAcaseA) was cloned using PCR with degenerate oligonucleotide primers from the partial amino acid sequence of the enzyme. The gene encoded a polypeptide of 863 amino acids with a predicted molecular mass of 97kDa. A characteristic signal peptide, which was present at the amino-terminus of the precursor protein, contained four amino acids (Ala-Gly-Cys-Ser) identical in sequence and location to the processing and modification sites of the outer membrane lipoprotein of Escherichia coli, indicating that the mature GlcNAcaseA is a lipoprotein the N-terminal cysteine residue of which would be modified by the fatty acid that anchors the protein in the membrane. The predicted amino acid sequence of GlcNAcaseA showed similarity to bacterial beta-N-acetylglucosaminidases belonging to the family 20 glycosyl hydrolases.     

Primary structure and processing of the Candida tsukubaensis alpha-glucosidase. Homology with the rabbit intestinal sucrase-isomaltase complex and human lysosomal alpha-glucosidase.

The nucleotide sequence of a 4.39-kb DNA fragment encoding the alpha-glucosidase gene of Candida tsukubaensis is reported. The cloned gene contains a major open reading frame (ORF 1) which encodes the alpha-glucosidase as a single precursor polypeptide of 1070 amino acids with a predicted molecular mass of 119 kDa. N-terminal amino acid sequence analysis of the individual subunits of the purified enzyme, expressed in the recombinant host Saccharomyces cerevisiae, confirmed that the alpha-glucosidase precursor is proteolytically processed by removal of an N-terminal signal peptide to yield the two peptide subunits 1 and 2, of molecular masses 63-65 kDa and 50-52 kDa, respectively. Both subunits are secreted by the heterologous host S. cerevisiae in a glycosylated form. Coincident with its efficient expression in the heterologous host, the C. tsukubaensis alpha-glucosidase gene contains many of the canonical features of highly expressed S. cerevisiae genes. There is considerable sequence similarity between C. tsukubaensis alpha-glucosidase, the rabbit sucrase-isomaltase complex (proSI) and human lysosomal acid alpha-glucosidase. The cloned DNA fragment from C. tsukubaensis contains a second open reading frame (ORF 2) which has the capacity to encode a polypeptide of 170 amino acids. The function and identity of the polypeptide encoded by ORF 2 is not known.     

Nucleotide sequence of the Synechococcus sp. PCC7942 branching enzyme gene (glgB): expression in Bacillus subtilis

The nucleotide sequence of the Synechococcus sp. PCC7942 glgB gene has been determined. The gene contains a single open reading frame (ORF) of 2322 bp encoding a polypeptide of 774 amino acids (aa) with an Mr of 89,206. Extensive sequence similarity exists between the deduced aa sequence of the Synechococcus sp. glgB gene product and that of the Escherichia coli branching enzyme in the middle portions of the proteins (62% identical aa). In contrast, the N-terminal portions shared little homology. The sequenced region which follows glgB contains an ORF encoding 79 aa of the N terminus of a polypeptide that shares extensive sequence similarity (41% identical aa) with human and rat uroporphyrinogen decarboxylase. This suggests that the region downstream from glgB contains the hemE gene and, therefore, that the organization of genes involved in glycogen biosynthesis in Synechococcus sp. is different from that described for E. coli. A fusion gene was constructed between the 5' end of the Bacillus licheniformis penP gene and the Synechococcus sp. glgB gene. The fusion gene was efficiently expressed in the Gram+ micro-organism Bacillus subtilis and specified a branching enzyme with an optimal temperature for activity similar to the wild-type enzyme.