The reactive site of marinostatin, a proteinase inhibitor from marine Alteromonas sp. B-10-31.

A new homologue of marinostatin, a peptide proteinase inhibitor, was isolated from marine Alteromonas sp. B-10-31 and designated as marinostatin D. Its amino acid sequence was determined to be Ala-Thr-Met-Arg-Tyr-Pro-Ser-Asp-Asp-Ser-Glu. The reactive site of marinostatin D was determined to be Met(3)-Arg(4) on the basis of the reversible cleavage and regeneration of the scissile bond catalyzed by TLCK-chymotrypsin.     

Structure‐activity relationship studies of shortened analogues of the antimicrobial peptide EeCentrocin 1 from the sea urchin Echinus esculentus

EeCentrocin 1 is a potent antimicrobial peptide isolated from the marine sea urchin Echinus esculentus. The peptide has a hetero‐dimeric structure with the antimicrobial activity confined in its largest monomer, the heavy chain (HC), encompassing 30 amino acid residues. The aim of the present study was to develop a shorter drug lead peptide using the heavy chain of EeCentrocin 1 as a starting scaffold and to perform a structure‐activity relationship study with sequence modifications to optimize antimicrobial activity. The experiments consisted of 1) truncation of the heavy chain, 2) replacement of amino acids unfavourable for in vitro antimicrobial activity, and 3) an alanine scan experiment on the truncated and modified heavy chain sequence to identify essential residues for antimicrobial activity. The heavy chain of EeCentrocin 1 was truncated to less than half its initial size, retaining most of its original antimicrobial activity. The truncated and optimized lead peptide ( P6 ) consisted of the 12 N‐terminal amino acid residues from the original EeCentrocin 1 HC sequence and was modified by two amino acid replacements and a C‐terminal amidation. Results from the alanine scan indicated that the generated lead peptide ( P6 ) contained the optimal sequence for antibacterial activity, in which none of the alanine scan peptides could surpass its antimicrobial activity. The lead peptide ( P6 ) was also superior in antifungal activity compared to the other peptides prepared and showed minimal inhibitory concentrations (MICs) in the low micromolar range. In addition, the lead peptide ( P6 ) displayed minor haemolytic and no cytotoxic activity, making it a promising lead for further antimicrobial drug development.     

Production of a recombinant chitin oligosaccharide deacetylase from Vibrio parahaemolyticus in the culture medium of Escherichia coli cells

An open reading frame (ORF) encoding chitin oligosaccharide deacetylase (Pa-COD) gene and its signal sequence was cloned from the Vibrio parahaemolyticus KN1699 genome and its sequence was analyzed. The ORF encoded a 427 amino acid protein, including the 22 amino acid signal sequence. The deduced amino acid sequence was highly similar to several bacterial chitin oligosaccharide deacetylases in carbohydrate esterase family 4. An expression plasmid containing the gene was constructed and inserted into Escherichia coli cells and the recombinant enzyme was secreted into the culture medium with the aid of the signal peptide. The concentration of the recombinant enzyme in the E. coli culture medium was 150 times larger than that of wild-type enzyme produced in the culture medium by V. parahaemolyticus KN1699. The recombinant enzyme was purified to homogeneity from culture supernatant in an overall yield of 16%. Substrate specificities of the wild-type and the recombinant enzymes were comparable.     

粘质沙雷氏菌几丁质酶基因ChiA克隆及生物信息学分析

通过生物信息学技术对Chi A基因序列进行分析预测,了解Chi A的基因结构及蛋白质性质。从自有菌株(粘质沙雷氏菌Serratia mareescens S68)中克隆到几丁质酶基因Chi A,利用相关软件对Chi A基因序列进行分析预测。Chi A基因全长1 714 bp,开放阅读框编码563个氨基酸,推测其编码的蛋白质分子量为60 983.8Da,等电点为6.35,是一种稳定的亲水性蛋白质。预测Chi A可能存在信号肽,切割位点在第23~24位氨基酸之间,1~23位氨基酸为其跨膜结构,其余肽链位于细胞外。Chi A主要存在3种二级结构元件,在二级、三级结构中都有体现。该Chi A是一种水溶性蛋白质,结构稳定且可以分泌到胞外。     

Primary structure of a pheromone-binding protein from Antheraea pernyi: homologies with other ligand-carrying proteins

Summary An antennal cDNA clone encoding the complete sequence (163 amino acids) of a pheromone-binding protein precursor from the male silk moth, Antheraea pernyi, was isolated using oligonucleotide probes. The cloned cDNA was expressed and the translation product detected by specific antibodies. The deduced protein sequence consists of a signal peptide of 21 amino acids and a mature binding protein of 142 amino acid residues. The predicted structure of this protein is homologous to binding-proteins from different insect species which have previously been identified, but shows no similarities to odorant-binding proteins from vertebrates, suggesting that soluble odorant-binding proteins in insects and vertebrates represent an evolutionary convergence.Abbreviations PBP pheromone-binding protein - OBP odorant-binding protein - cDNA complentary DNA - poly(A ⁺) RNA polyadenylated RNA - SSC 0.15 M sodium chloride+0.015 M sodium citrate - SDS-PAGE sodium dodecylsulfate polyacrylamide gelelectrophoresis     

Sequence analysis of the gene encoding a novel l-2,4-diaminobutyrate decarboxylase of Acinetobacter baumannii: similarity to the group II amino acid decarboxylases

The gene (ddc) encoding a novel enzyme, l-2,4-diaminobutyrate decarboxylase (DABA-DC; EC 4.1.1.-) in Acinetobacter baumannii was sequenced, and an open reading frame of 1,530 nucleotides was detected. The sequence of 20 N-terminal amino acids of purified DABA-DC and of its proteolytic peptide fragments coincided with those deduced from the nucleotide sequence determined. Comparison of the predicted amino acid sequence of the A. baumannii enzyme with those of other pyridoxal 5′-phosphate-dependent decarboxylases revealed significant similarity to the group II amino acid decarboxylases and conservation of the putative pyridoxal 5′-phosphate-binding domain. Received:20 February 1996 / Accepted 15 April 1996     

Sequence of the regulatory region of omp T, the gene specifying major outer membrane protein a (3b) of Escherichia coli K-12: implications for regulation and processing

Summary The DNA of the promoter region of ompT, including the putative start for the pro-OmpT protein (proprotein a), has been sequenced. Previous studies showed that trypsin inhibitors prevent the processing of pro-OmpT to OmpT protein which led to the prediction that the processing site would be a lysine or an arginine. The deduced amino acid sequence contains a lysine at amino acid 12 and an arginine at amino acid 17 from the N terminus. Chou-Fassman analysis would predict processing at the lysine (but not the arginine) to remove a 1389 dalton peptide, consistent with the fact that the estimated molecular masses of pro-OmpT and OmpT are 42 kd and 40 kd respectively. In addition, the predicted mRNA of the promoter region can form a stable secondary structure (-17.1 kcal) that sequesters the Shine-Dalgarno (SD) sequence as well as the initiator AUG codon. There is evidence that the perA (tpo, envZ) gene product is required for synthesis of OmpT protein (as well as several outer membrane and periplasmic proteins). The perA gene product could be activating translation of OmpT protein by disrupting the mRNA secondary structure that sequesters the SD sequence. OmpT protein synthesis is reduced at temperatures below 32°C and this may also be related to the greater stability of the sequestered SD sequence of the mRNA at low temperature.     

The <Emphasis Type="Italic">Chlamydomonas reinhardtii gtr </Emphasis>Gene Encoding the Tetrapyrrole Biosynthetic Enzyme Glutamyl-tRNA Reductase: Structure of the Gene and Properties of the Expressed Enzyme

Plants, algae, cyanobacteria and many other bacteria synthesize the tetrapyrrole precursor, δ-aminolevulinic acid (ALA), from glutamate by means of a tRNA^Glu-mediated pathway. The enzyme glutamyl-tRNA reductase (GTR) catalyzes the first committed step in this pathway, which is the reduction of tRNA-bound glutamate to produce glutamate 1-semialdehyde. Chlamydomonas reinhardtii mRNA encoding gtr was sequenced from a cDNA and genomic libraries. The 3179-bp gtr cDNA contains a 1566-bp open reading frame that encodes a 522-amino acid polypeptide. After removal of the predicted transit peptide, the mature 480-residue GTR has a calculated molecular weight of 52,502. The deduced C. reinhardtii mature GTR amino acid sequence has more than 55% identity to a GTR sequence of Arabidopsis thaliana, and significant similarity to GTR proteins of other plants and prokaryotes. Southern blot analysis of C. reinhardtii genomic DNA indicates that C. reinhardtii has only one gtr gene. Genomic DNA sequencing revealed the presence of a small intron near the putative transit peptide cleavage site. Expression constructs for the full-length initial gtr translation product, the mature protein after transit peptide removal, and the coding sequence of the second exon were cloned into expression vector that also introduced a C-terminal His₆ tag. All of these constructs were expressed in E. coli, and both the mature protein and the exon 2 translation product complemented a hemA mutation. The expressed proteins were purified by Ni-affinity column chromatography to yield active GTR. Purified mature GTR was not inhibited by heme, but heme inhibition was restored upon addition of C. reinhardtii soluble proteins.     

Cloning and Sequence Analysis of a Protease-encoding Gene from the Marine Bacterium Alteromonas sp. Strain O-7

The gene (aprI) encoding alkaline serine protease (AprI; subtilase) from Alteromonas sp. strain O-7 was cloned and sequenced. The nucleotide sequence of aprI has been identified. The deduced amino acid sequence indicated that aprI codes for a precursor of 715 amino acids and the precursor is composed of four regions including a signal peptide, an N-terminal pro-region, a mature protease region and a C-terminal extension region of 215 amino acids as previously described for aprII [H. Tsujibo et al., Gene, 136, 247–251 (1993)]. The amino acid sequence of the mature AprI (AprI-M) showed high sequence homology with those of other class I subtilases. The C-terminal region was characterized by a repeat of 94 amino acids residues, which showed about 50% similarity with those of the C-terminal pro-region of several known proteases from Gram-negative bacteria.     

Cloning and expression of a gene for an alpha-glucosidase from Saccharomycopsis fibuligera homologous to family GH31 of yeast glucoamylases

Cloning of cDNA encoding an α-glucosidase from the dimorphous yeast Saccharomycopsis fibuligera and characterization of the gene product were performed. The cDNA of the putative α-glucosidase gene consists of 2,886 bp, which includes an open reading frame encoding a 19 amino acid signal peptide at the N-terminal end and a 944 amino acid mature protein with a predicted molecular mass of 105.4 kDa and pI value of 4.52. The deduced amino acid sequence shows a high degree of identity (70%) with two yeast glucoamylases, namely, the extracellular glucoamylase Gam from Schwanniomyces occidentalis and the cell surface glucoamylase Gca from Candida albicans. The recombinant product, synthesized in Saccharomyces cerevisiae, is localized on the cell surface and hydrolyses maltooligosaccharides exclusively without the ability to digest soluble starch, which is consistent with the specificity characteristic of α-glucosidase, EC. 3.2.1.20.     

Style-specific and developmentally regulated accumulation of a glycosylated thaumatin/PR5-like protein in Japanese pear (Pyrus serotina Rehd.)

The stylar proteins of Japanese pear (Pyrus serotina Rehd.) were analyzed by two-dimensional gel electrophoresis, and a 32-kDa protein with an isoelectric point of 4.8 was found to be a major component in the style. The 32-kDa protein was a soluble glycoprotein which reacted with concanavalin A. The 32-kDa protein specifically accumulated in the style in a developmentally regulated manner, but was not detected in the other floral organs and leaves. An oligonucleotide representing the N-terminal amino acid sequence of the 32-kDa protein was used to amplify a cDNA fragment by polymerase chain reaction (PCR). The generated PCR product was used to screen a style cDNA library. The selected cDNA clone encoded 244 amino acid residues containing the N-terminal sequence of the 32-kDa protein. The N-terminus of the protein was preceded by putative signal peptide of 22 amino acid residues. The 32-kDa protein showed significant homology with the thaumatin/PR5-like proteins, and was named PsTL1 (Pyrus serotina thaumatin-like protein 1). The possible biological role of PsTL1 in the styles is discussed. Received: 27 November 1997 / Accepted: 19 January 1998     

Identification of preferred substrate sequences of microbial transglutaminase from Streptomyces mobaraensis using a phage-displayed peptide library

Microbial transglutaminase (TGase) from Streptomyces mobaraensis (MTG) has been used in many industrial applications because it effectively catalyzes the formation of covalent cross-linking between glutamine residues in various substrate proteins and lysine residues or primary amines. To better understand the sequence preference around the reactive glutamine residue by this enzymatic reaction, we screened preferred peptide sequences using a phage-displayed random peptide library. Most of the peptides identified contained a consensus sequence, which was different from those previously found for mammalian TGases. Of these, most sequences had a specific reactivity toward MTG when produced as a fusion protein with glutathione-S-transferase. Furthermore, the representative sequence was found to be reactive even in the peptide form. The amino acid residues in the sequence critical for the reactivity were further analyzed, and the possible interaction with the enzyme has been discussed in this paper.     

Identification and expression of a cDNA from Daucus carota encoding a bifunctional aspartokinase-homoserine dehydrogenase

Aspartokinase (EC 2.7.2.4) and homoserine dehydrogenase (EC 1.1.1.3) catalyze steps in the pathway for the synthesis of lysine, threonine, and methionine from aspartate. Homoserine dehydrogenase was purified from carrot (Daucus carota L.) cell cultures and portions of it were subjected to amino acid sequencing. Oligonucleotides deduced from the amino acid sequences were used as primers in a polymerase chain reaction to amplify a DNA fragment using DNA derived from carrot cell culture mRNA as template. The amplification product was radiolabelled and used as a probe to identify cDNA clones from libraries derived from carrot cell culture and root RNA. Two overlapping clones were isolated. Together the cDNA clones delineate a 3089 bp long sequence encompassing an open reading frame encoding 921 amino acids, including the mature protein and a long chloroplast transit peptide. The deduced amino acid sequence has high homology with the Escherichia coli proteins aspartokinase I-homoserine dehydrogenase I and aspartokinase II-homoserine dehydrogenase II. Like the E. coli genes the isolated carrot cDNA appears to encode a bifunctional aspartokinase-homoserine dehydrogenase enzyme.Abbreviations AK aspartokinase - HSDH homoserine dehydrogenase - PCR polymerase chain reaction - SDS sodium dodecyl sulfate The mention of vendor or product does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over vendors of similar products not mentioned.     

Isolation and Characterization of a Carbonic Anhydrase Homologue from the Zebrafish (Danio rerio)

We have isolated a 29,000-Da carbonic anhydrase (CA) protein from the zebrafish, Danio rerio, sequenced two peptide fragments, and tentatively identified it as a high-activity CA by inhibition kinetics. We have also characterized a 1,537-bp message whose deduced sequence of 260 amino acids matches that of the isolated protein. This CA is clearly an α-CA based on the similarity of its sequence to that of other members of the α-CA gene family. A phylogenetic analysis suggested CAH-Z diverged after the branching of the CA-V and CA-VII genes and prior to the duplications that generated the CA-I, CA-II, and CA-III genes of amniotes. This marks the first characterization of the mRNA and its protein product from the CA gene of a teleost. Received: 31 March 1996 / Accepted: 8 September 1996     

The CFTR-derived peptides as a model of sequence-specific protein aggregation

Protein aggregation is a hallmark of a growing group of pathologies known as conformational diseases. Although many native or mutated proteins are able to form aggregates, the exact amino acid sequences involved in the process of aggregation are known only in a few cases. Hence, there is a need for different model systems to expand our knowledge in this area. The so-called ag region was previously found to cause the aggregation of the C-terminal fragment of the cystic fibrosis transmembrane conductance regulator (CFTR). To investigate whether this specific amino acid sequence is able to induce protein aggregation irrespective of the amino acid context, we altered its position within the CFTR-derived C-terminal peptide and analyzed the localization of such modified peptides in transfected mammalian cells. Insertion of the ag region into a different amino acid background affected not only the overall level of intracellular protein aggregation, but also the morphology and subcellular localization of aggregates, suggesting that sequences other than the ag region can substantially influence the peptide’s behavior. Also, the introduction of a short dipeptide (His-Arg) motif, a crucial component of the ag region, into different locations within the C-terminus of CFTR lead to changes in the aggregation pattern that were less striking, although still statistically significant. Thus, our results indicate that even subtle alterations within the aggregating peptide can affect many different aspects of the aggregation process.     

Isolation of a cDNA clone for the acyl carrier protein-I of spinach

A 715 base pair cDNA clone coding for an acyl carrier protein (ACP) in spinach leaves has been isolated and characterized. The amino acid sequence indicated by the cDNA sequence closely matches the amino acid sequence of the ACP-I isoform. The presence of polyadenylation and DNA sequence coding for a precursor protein with a putative transit peptide, and the absence of hybridization between the cloned DNA and isolated spinach plastid DNA collectively show that the ACP-I gene is nuclear-encoded. The ACP-I cloned DNA did not cross-hybridize with mRNA from spinach tissues in which ACP-II has been found. Cross-hybridization with mRNA from tissues of Brassica campestris was either weak or undetectable. The cloning of an ACP-I gene represents an initial step in the molecular dissection of fatty acid synthetase in plants.