The structure of subtilisin ALP I from alkalophilic Bacillus sp. NKS-21

The gene for an alkaline serine protease from alkalophilic Bacillus sp. NKS-21 (subtilisin ALP I) was cloned, and its nucleotide sequence was determined. The gene (aprQ) contained an open reading frame of 1125 bp, encoding a primary product of 374 amino acids. The mature protease, composed of 272 amino acids, was preceded by a putative signal sequence of 37 amino acids and a pro-sequence of 65 amino acids. The mature protease conserved the catalytic triad, Asp, His, and Ser, as subtilisin BPN or other subtilisins, and the subtilisin ALP I might belong to the subtilisin super family. The primary structure of subtilisin ALP I was compared and discussed with those of 13 subtilisins, 5 subtilisins from alkalophilic Bacillus, and 8 from neutrophiles. Low homology was shown between subtilisin ALP I and subtilisins from alkalophiles or subtilisins from neutrophiles. Forty-five amino acid residues of the mature protein of subtilisin ALP I were entirely independent of other subtilisins. According to the homology of ALP I with other subtilisins, subtilisin ALP I might be in the middle point between alkaline subtilisins and neutral ones.     

Molecular characterization of the Oncidium orchid HDR gene encoding 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase, the last step of the methylerythritol phosphate pathway

Two pathways are used by higher plants for the biosynthesis of isoprenoid precursors: the mevalonate pathway in the cytosol and a 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway in the plastids, with 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR) catalyzing the last step in the MEP pathway. In order to understand the contribution of MEP pathway in isoprenoid biosynthesis of Oncidium orchid, a full-length cDNA corresponding to HDR from the flower tissues of Oncidium Gower Ramsey was cloned. The deduced OncHDR amino acid sequence contains a plastid signal peptide at the N-terminus and four conserved cysteine residues. RT-PCR analysis of HDR in Oncidium flowering plants revealed ubiquitous expression in organs and tissues, with preferential expression in the floral organs. Phylogenetic analysis revealed evolutionary conservation of the encoding HDR protein sequence. The genomic sequence of the HDR in Oncidium is similar to that in Arabidopsis, grape, and rice in structure. Successful complementation by OncHDR of an E. coli hdr ⁻ mutant confirmed its function. Transgenic tobacco carrying the OncHDR promoter-GUS gene fusion showed expression in most tissues, as well as in reproductive organs, as revealed by histochemical staining. Light induced strong GUS expression driven by the OncHDR promoter in transgenic tobacco seedlings. Taken together, our data suggest a role for OncHDR as a light-activated gene.     

A proline-rich chitinase from Beta vulgaris

A gene (Chl) encoding a novel type of chitinase was isolated from Beta vulgaris. The Ch1 protein consists of an N-terminal hydrophobic prepeptide of 25 amino acids followed by a hevein-like domain of 22 amino acid residues, an unusually long proline-rich domain of 131 amino acid residues with 90 prolines, and finally a catalytic domain of 261 amino acid residues. Proteins with similar proline-rich domains are present in some other plants. The Chl gene shows a transient expression in response to fungal infection.     

Cloning and Sequence Analysis of cDNA encoding Rhizopus niveus Lipase

Complementary DNA encoding Rhizopus niveus lipase (RNL) was isolated from the R. niveus IF04759 cDNA library using a synthetic oligonucleotide corresponding to the amino acid sequence of the enzyme. A clone, which had an insert of 1.0 kilobase pairs, was found to contain the coding region of the enzyme. The lipase gene was expressed in Escherichia coli as a lacZ fusion protein. The mature RNL consisted of 297 amino acid residues with a molecular mass of 32 kDa. The RNL sequence showed significant overall homology to Rhizomucor miehei lipase and the putative active site residues were strictly conserved.     

Molecular analysis of the Aureobasidium pullulans ura3 gene encoding orotidine-5′-phosphate decarboxylase and isolation of mutants defective in this gene

Orotidine-5′-phosphate decarboxylase (OMP decarboxylase) catalyses the final step in the pyrimidine biosynthesis, the conversion of orotidine-5′-phosphate (OMP) to uridine-5′-phosphate. The ura3 gene of Aureobasidium pullulans, encoding OMP decarboxylase, was isolated from an Aureobasidium genomic library constructed in the plasmid pBlueskriptSK⁻. The ura3 gene of A. pullulans has an open reading frame of 271 amino acid residues. Analysis of the sequence revealed the presence of two introns. In the predicted amino acid sequence there are regions of strong homology to the equivalent genes of Aspergillus niger, Neurospora crassa, Phycomyces blakesleeanus and Homo sapiens. The ura3 gene is the third Aureobasidium gene that has been cloned and analysed. We have also isolated ura3 mutants by selection of ethyl methanesulphonate mutagenised cells on 5-fluoroorotic acid. Transformation of these A. pullulans mutant strains to prototrophy showed the functionality of the cloned gene. Received: 16 July 1999 / Revision received: 20 September 1999 / Accepted: 24 September 1999     

Tests of the ribosome editor hypothesis. II. Relaxed (relA) and stringent (relA+) E. coli differ in rates of dissociation of peptidyl-tRNA from ribosomes

Summary Two mutants of Escherichia coli that are deficient in the penicillin-insensitive DD-endopeptidase have been isolated. The strain JE10874 (mepA) has about 10%–20% of the residual activity and another strain, JE10368 (mepB) has 40%–50% of the activity found in the wild-type, parental strain, PA3092. The penicillin-insensitive endopeptidase is a periplasmic enzyme. Genetic mapping studies show that the mutation mepA is located close to aroC (50 min) and the other mutation, mepB, is very close to malE (91 min) on the chromosome. These mutants grow normally under a wide range of growth conditions; other phenotypic properties of the mutants are very similar to those of the parent strain. A double mutant (mepA mepB), and a triple mutant (mepA mepB dacB), deficient in both penicillin-insensitive and penicillin-sensitive endopeptidases, were constructed. Again, these mutants grew normally. We conclude that either the very low level of residual enzyme activity in the mutants is enough for their survival or that the penicillin-insensitive endopeptidase is not essential for survival under laboratory conditions.     

Deletion of the <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> lysine biosynthesis gene <Emphasis Type="Italic">lysF</Emphasis> encoding homoaconitase leads to attenuated virulence in a low-dose mouse infection model of invasive aspergillosis

Aspergillus fumigatus is an important pathogen of the immunocompromised host, causing pneumonia and invasive disseminated disease with high mortality. In order to determine the importance of lysine biosynthesis for growth and pathogenicity, the A. fumigatus lysF gene, encoding a homologue of the A. nidulans homoaconitase LysF, was cloned and characterized. Cosmid cosGTM encoding lysF complemented a lysF mutant of Aspergillus nidulans. A. fumigatus lysF was deleted, resulting in a lysine-auxotroph. This phenotype was complemented to the wild-type by supplementation of the medium with both L-lysine and -aminoadipic acid, or transformation using cosmid cosGTM. To study the virulence of the lysF deletion mutant of A. fumigatus, a low-dose intranasal mouse infection model of invasive aspergillosis was optimized for immunosuppressed BALB/c mice, allowing the application of an infection dose as low as 5×10³ conidia per mouse. In this murine model, the lysF mutant was avirulent, suggesting that lysine biosynthesis, or at least a functional homoaconitase, is important for survival of A. fumigatus in vivo and a potential target for antifungal drugs.     

Cloning,characterization and expression of the extracellular lipase gene from <Emphasis Type="Italic">Aureobasidium </Emphasis><Emphasis Type="Italic">pullulans</Emphasis> HN2-3 isolated from sea saltern

The extracellular lipase structural gene was isolated from cDNA of Aureobasidium pullulans HN2-3 by using SMART^TM RACE cDNA amplification kit. The gene had an open reading frame of 1245 bp long encoding a lipase. The coding region of the gene was interrupted by only one intron (55 bp). It encodes 414 amino acid residues of a protein with a putative signal peptide of 26 amino acids. The protein sequence deduced from the extracellular lipase structural gene contained the lipase consensus sequence (G-X-S-X-G) and three conserved putative N-glycosylation sites. According to the phylogenetic tree of the lipases, the lipase from A. pullulans was closely related to that from Aspergillus fumigatus (XP_750543) and Neosartorya fischeri (XP_001257768) and the identities were 50% and 52%, respectively. The mature peptide encoding cDNA was subcloned into pET-24a (+) expression vector. The recombinant plasmid was expressed in Escherichia coli BL21(DE3). The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 47 kDa was found. Enzyme activity assay verified the recombinant protein as a lipase. A maximum activity of 0.96 U/mg was obtained from cellular extract of E. coli BL21(DE3) harboring pET-24a(+)LIP1. Optimal pH and temperature of the crude recombinant lipase were 8.0 and 35 °C, respectively and the crude recombinant lipase had the highest hydrolytic activity towards peanut oil.     

Probing the active site of mitogillin,a fungal ribotoxin

Fungal ribotoxins, such as mitogillin and the related Aspergillus toxins restrictocin and α-sarcin, are highly specific ribonucleases, which inactivate the ribosome enzymatically by cleaving the eukaryotic 28S RNA of the large ribosomal subunit at a single phosphodiester bond. The site of cleavage occurs between G₄₃₂₅ and A₄₃₂₆, which are present in a 14-base sequence (the α-sarcin loop) conserved among the large subunit rRNAs of all living species. The amino acid residues involved in the cytotoxic activities of mitogillin were investigated by introducing point mutations using hydroxylamine into a recombinant Met-mature mitogillin (mitogillin with a Met codon at the N-terminus and no leader sequence) gene constructed from an Aspergillus fumigatus cDNA clone. These constructs were cloned into a yeast expression vector under the control of the GAL1 promoter and transformed into Saccharomyces cerevisiae. Upon induction of mitogillin expression, surviving transformants revealed that substitutions of certain amino acid residues on mitogillin abolished its cytotoxicity. Non-toxic mutant genes were cloned into an Escherichia coli expression vector, the proteins overexpressed and purified to homogeneity and their activities examined by in vitro ribonucleolytic assays. These studies identified the His-49Tyr, Glu-95Lys, Arg-120Lys and His-136Tyr mutations to have a profound impact on the ribonucleolytic activities of mitogillin. We conclude that these residues are key components of the active site contributing to the catalytic activities of mitogillin.     

Cloning and sequence analysis of theMaackia amurensis haemagglutinin cDNA

Maackia amurensis haemagglutinin (MAH) is a leguminous lectin which preferentially binds to a cluster of sialylatedO-linked carbohydrate chains (Konami Y, Yamamoto K, Osawa T, Irimura T (1994)FEBS Lett 342:334–38). In the present study a 950 bp cDNA clone encoding MAH was isolated from a cDNA library constructed from germinatedMaackia amurensis seeds. From the nucleotide sequence, MAH was predicted to consist of 285 amino acid residues containing a signal peptide of 29 amino acids. The results also confirmed our previous findings from the amino acid sequence analysis, which indicated that two highly conserved amino acid residues in all other well-known leguminous lectins were replaced in MAH. These residues were lysine-105 and aspartic acid-135. The corresponding amino acid residues in other leguminous lectins were glycine and asparagine, respectively. These differences were due to the presence of nucleotides AAA and GAT in place of AAT/C and GGA/T.Abbreviations MAH Maackia amurensis haemagglutinin.     

Stereoselective synthesis of (<Emphasis Type="Italic">R</Emphasis>)-3-quinuclidinol through asymmetric reduction of 3-quinuclidinone with 3-quinuclidinone reductase of <Emphasis Type="Italic">Rhodotorula rubra</Emphasis>

A novel nicotinamide adenine dinucleotide phosphate-dependent carbonyl reductase, 3-quinuclidinone reductase, was isolated from Rhodotorula rubra JCM3782. The enzyme catalyzes the asymmetric reduction of 3-quinuclidinone to (R)-3-quinuclidinol. The gene encoding the enzyme was also cloned and sequenced. A 819-bp nucleotide fragment was confirmed to be the gene encoding the 3-quinuclidinone reductase by agreement of the internal amino acid sequences of the purified enzyme. The gene encodes a total of 272 amino acid residues, and the deduced amino acid sequence shows similarity to those of several short-chain dehydrogenase/reductase family proteins. An expression vector, pWKLQ, which contains the full length 3-quinuclidinone reductase gene was constructed. Using Escherichia coli cells coexpressing the 3-quinuclidinone reductase and glucose dehydrogenase (cofactor regeneration enzyme) genes, 618 mM 3-quinuclidinone was almost stiochiometrically converted to (R)-3-quinuclidinol with an >99.9% enantiomeric excess within 21 h of reaction.     

A Corynebacterium glutamicum rnhA recG Double Mutant Showing Lysozyme- sensitivity,Temperature-sensitive Growth,and UV-Sensitivity

Corynebacterium glutamicum mutant KY9707 was originally isolated for lysozyme-sensitivity, and showed temperature-sensitive growth. Two DNA fragments from a wild-type C. glutamicum chromosomal library suppressed the temperature-sensitivity of KY9707. These clones also rescued the lysozyme-sensitivity of KY9707, although partially. One of them encodes a protein of 382 amino acid residues, the N-terminal domain of which was homologous to RNase HI. This gene suppressed the temperature-sensitive growth of an Escherichia coli rnhA rnhB double mutant. We concluded that this gene encodes a functional RNase HI of C. glutamicum and designated it as rnhA. The other gene encodes a protein of 707 amino acid residues highly homologous to RecG protein. The C. glutamicum recG gene complemented the UV-sensitivity of E. coli recG258::kan mutant. KY9707 showed increased UV-sensitivity, which was partially rescued by either the recG or rnhA gene of C. gluamicum. Point mutations were found in both recG and rnhA genes in KY9707. These suggest that temperature-sensitive growth, UV-sensitivity, and probably lysozyme-sensitivity also, of KY9707 were caused by mutations in the genes encoding RNase HI and RecG.     

Purification of Cytochrome P-450«/A from n-Alkane-grown Cells of Candida maltosa,and Cloning and Nucleotide Sequencing of the Encoding Gene

When the cells of an n-alkane-assimilating yeast, Candida maltosa I AM12247, were transferred from a glucose medium to an n-alkane medium, various enzymes are induced in the endoplasmic reticulum and peroxisome. Cytochrome P-450alk, one of these enzymes in the endoplasmic reticulum, was purified after mild solubilization of the membrane, followed by a few steps of chromatography. The enzyme was characterized spectrophotometrically and its N-terminal amino acid sequence (12 residues) was determined.

Using oligonucleotide probes prepared to match parts of the N-terminal amino acid sequence and of the partial cDNA sequence of cytochrome P-450alk of C. maltosa EH 15, we isolated from a gene library of C. maltosa I AM 12247 a clone which had a gene encoding cytochrome P-450a/Ar. By nucleotide sequencing of this gene, the amino acid sequence of this enzyme was deduced. It consisted of 523 amino acids (59,838 daltons), with a non-cleavable signal sequence in the N-terminal region. The structure of this enzyme was compared with some other members of the cytochrome P-450 superfamily.     

Molecular cloning and analysis of a cDNA coding for nucleoside diphosphate kinase from ryegrass

A full-length cDNA, LpNDPK, encoding ryegrass nucleoside diphosphate kinase (EC 2.7.4.6) has been cloned and sequenced. The nucleotide sequence of the clone contains an open reading frame of 450 nucleotides encoding a protein of 150 amino acid residues with a calculated molecular mass of 16.5 kDa and a P_i of 6.62. The LpNDPK encoded protein possesses substantial homology with nucleoside diphosphate kinases (NDPKs) isolated and cloned form other sources; the highest identity (86 percnt;) was observed with NDPK from sugarcane (Saccharum officinarum). Amino acid comparisons with other NDPKs show that the presented ryegrass NDPK sequence also contains several motifs and specific residues crucial for catalytic activity which are highly conserved among other NDPKs. RT-PCR expression analysis using primers covering the coding region of LpNDPK revealed that the ryegrass NDPK gene is equally expressed in stem, leaf, and flower tissue.     

Purification,Characterization and Gene Analysis of N-Acetylglucosaminidase from Enterobacter sp. G-1

Enterobacter sp. G-1 is a bacterium isolated previously as a chitinase-producing bacterium. We found this bacterium also produced N-acetylglucosaminidase and characterized that in this study. Extracellular N-acetylglucosaminidase of 92.0 kDa was purified near homogeneity by 8.57-fold from Enterobacter sp. G-1. The optimum temperature and the optimum pH of the purified N-acetylglucosaminidase was 45°C and 6.0, respectively. The N-terminal amino acid sequence of 23 residues of N-acetylglucosaminidase was identified. Based on the N-terminal sequence, we amplified pieces of the DNA fragments by PCR. Using these PCR products as probes, we screened the genomic library and successfully isolated the entire N-acetyl-glucosaminidase gene (designated nag1) from Enterobacter sp. G-1. The nucleotide sequence of the nag1 gene was found to consist of 2,655 bp encoding a protein of 885 amino acid residues. Comparison of the deduced amino acid sequence from the nag1 gene found 97.3% identity with chitobiase from Serratia marcescens, 54.4% identity with N,N′-diacetylchitobiase from Vibrio harveyi, and 42.7% identity with N-acetylglucosaminidase (ExoI) from Vibrio furnissii. Enzymatic activity assay of N-acetylglucosaminidase indicated stronger activity toward PNP-GlcNAc than PNP-(GlcNAc)₂ or PNP-(GlcNAc)₃.     

<Emphasis Type="Italic">Aspergillus niger</Emphasis> lipase: gene cloning,over-expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> and in vitro refolding

From the N-terminal amino acid sequence of the lipase from Aspergillus niger F044, a potential homologous gene A84689 to the lipanl (the gene encoding the lipase from Aspergillus niger F044) was identified. A pair of primers were designed according to the nucleotide sequence of A84689, and the lipanl was cloned by PCR. Nucleotide sequencing revealed that the lipanl has an ORF of 1,044 bp, containing three introns. The deduced amino acid sequence corresponds to 297 amino acid residues. The cloned cDNA fragment encoding the mature lipase from Aspergillus niger F044 was over-expressed in Escherichia coli BL21(De3) and the recombinant protein was refolded in vitro by dilution followed by DEAE Sepharose Fast Flow chromatography.