Comparative amino acid sequence analysis of Thermotoga maritima β-glucosidase (BglA) deduced from the nucleotide sequence of the gene indicates distant relationship between β-glucosidases of the BGA family and other families of β-1,4-glycosyl hydrolases

The primary structure of the bglA gene region encoding a -glucosidase of Thermotoga maritima strain MSB8 was determined. The bglA gene has the potential to code for a polypeptide of 446 amino acids with a predicted molecular mass of 51545 Da. The T, maritima -glucosidase (BglA) was overexpressed in E. coli at a level comprising approximately 15–20% of soluble cellular protein. Based on its amino acid sequence, as deduced from the nucleotide sequence of the gene, BglA can be classified as a broad-specificity -glucosidase and as a member of the -glucosidase family BGA, in agreement with the results of enzymatic characterization of the recombinant protein. Comparative sequence analysis revealed distant amino acid sequence similarities between BGA family -glucosidases, a -xylosidase, -1,4-glycanases of the enzyme family F (mostly xylanases), and other families of -1,4-glycosyl hydrolases. This result indicates that BGA -glucosidases may comprise one enzyme family within a large enzyme order of retaining -glycosyl hydrolases, and that the members of these enzyme groups may be inter-related at the level of active site architecture and perhaps even on the level of overall three-dimensional fold.     

The nucleotide sequence of the β-glucosidase gene from Cellvibrio gilvus

A gene encoding β-glucosidase from Cellvibrio gilvus, a cellobiose-producing bacterium, was cloned into Escherichia coli and sequenced. The structural gene consisted of 2565 bp encoding 854 amino acid residues with a characteristic signal peptide. A typical promoter sequence and SD region were located upstream of the initiation ATG codon. A sequence (180 amino acids) having high homology with those of β-glucosidases from several microorganisms was found in the deduced amino acid sequence of C. gilvus β-glucosidase. This sequence contains the aspartic acid residue which was found to be an active site residue in Aspergillus wentii β-glucosidase A3. The β-glucosidase gene of C. gilvus contains a high amount (69.4%) of G+C. These bases are localized not in the 3rd position of the codon, as is usually observed in G+C-rich genes, but rather in the 1st position. This result in a peptide which contains an extremely high amount (48%) of four amino acids (Pro, Ala, Arg, Gly) coded by CCN, GCN, CGN, and GGN.     

Avenacosidase from oat: purification,sequence analysis and biochemical characterization of a new member of the BGA family of β-glucosidases

A protein consisting of 60 kDa subunits (As-P60) was isolated from etiolated oat seedlings (Avena sativa L.) and characterized as avenacosidase, a -glucosidase that belongs to a preformed defence system of oat against fungal infection. The enzyme is highly aggregated; it consists of 300–350 kDa aggregates and multimers thereof. Dissociation by freezing/thawing leads to complete loss of enzyme activity. The specificity of the enzyme was investigated with para-nitrophenyl derivatives which serve as substrates, in decreasing order -fucoside, -glucoside, -galactoside, -xyloside. The corresponding orthonitrophenyl glycosides are less well accepted. No hydrolysis was found with -glycosides and -thioglucoside. An anti-As-P60 antiserum was prepared and used for isolation of a cDNA clone coding for As-P60. A presequence of 55 amino acid residues was deduced from comparison of the cDNA sequence with the N-terminal sequence determined by Edman degradation of the mature protein. The presequence has the characteristics of a stroma-directing signal peptide; localization of As-P60 in plastids of oat seedlings was confirmed by western blotting. The amino acid sequence revealed significant homology (>39% sequence identity) to -glucosidases that are constituents of a defence mechanism in dicotyledonous plants. 34% sequence identity was even found with mammalian and bacterial -glucosidases of the BGA family. Avenacosidase extends the occurrence of this family of -glucosidases to monocotyledonous plants.     

Close phylogenetic relationship between vestimentifera (tube worms) and annelida revealed by the amino acid sequence of elongation factor-lα

To clarify the phylogenetic position of Vestimentifera (tube worms), 346-bp fragments of the elongation factor-l (EF-l) gene (939–1286 according to the numbering of the human gene) of a vestimentiferan, Lamellibrachia sp., a sternaspid polychaete, Sternaspis scutata, an earthworm, Pheretima sp., and a gastropod, Alviniconcha hessleri, were sequenced. From the amino acid sequences of these EF-l, and those of two other vertebrates and two arthropods, phylogenetic relationships were deduced by the maximum likelihood (ML) method, by which the phylogenetic tree can be inferred without assuming constancy of the molecular evolutionary rate. For the ML tree and all of seven alternative trees, whose log-likelihoods could not be discriminated from that of the ML tree by the criterion of the standard error, the vestimentiferan, the polychaete, and the oligochaete formed a clade, excluding the arthropods and the gastropod as outgroups. This result is convincing evidence that Vestimentifera are protostomes that are closely related to Annelida. The ML tree suggests that Vestimentifera are more closely related to Polychaeta than to Oligochaeta, though the data were not sufficient to discriminate these three groups at a significant level. From recent evidence such as morphological characteristics and molecular information, it may safely be said that vestimentiferans should be included in the Annelida provided this phylum contains polychaetes and oligochaetes.Correspondence to: S. Kojima     

Nucleotide and derived amino acid sequence of the cyanogenic β-glucosidase (linamarase) from white clover (Trifolium repens L.)

The nucleotide sequence and derived amino acid sequence of two different -glucosidase cDNA clones were determined. One clone (TRE104) was identified as the cyanogenic -glucosidase by homology with the N-terminal and internal peptide amino acid sequence of the purified enzyme. The biological function of the other -glycosidase (TRE361) is not known. Co-segregation of genomic restriction fragments uniquely identified by each cDNA clone shows that these two genes are linked in the white clover genome. Both TRE104 and TRE361 fragments co-segregate with cyanogenic -glucosidase activity. Extensive homology was found between the white clover -glucosidase sequences and a group of prokaryote and mammalian -glycosidases. This group of sequences has no homology with a separate set of -glucosidase genes isolated from fungi and the thermophilic bacterium Clostridium thermocellum.     

A phosphate-starvation inducible β-glucosidase gene (psr3.2) isolated from Arabidopsis thaliana is a member of a distinct subfamily of the BGA family

We have previously isolated a phosphate starvation-response (psr) cDNA clone, psr3.1, from Brassica nigra which encodes a -glucosidase. Southern blots of Arabidopsis thaliana genomic DNA probed with the psr3.1 cDNA indicated that this gene exists as a single locus. A genomic library of A. thaliana was screened at high stringency to isolate the corresponding genomic clone. The resultant clone was coined psr3.2 because of its sequence divergence from isolated psr3.1 cDNA clones. Northern blotting with probes derived from the coding region of the genomic clone showed that this gene is expressed at high levels in P_i-starved roots and the enhancement occurred within two days of growth in medium lacking P_i. The expression of this gene is repressed by heat shock and anaerobic conditions, and it is not significantly induced by high salinity, or by nitrogen or sulfur deprivation. Sequence analysis of the genomic clone revealed the existence of 13 exons interrupted by 12 AT-rich introns and it possessed a high homology with the B. nigra psr3.1 as well as various other -glucosidase genes from other species. Sequence similarity and divergence percentages between the deduced amino acid sequences of the psr3 clones and other -glycosidases suggests that they should be included along with two other Brassicaceae genes in a distinct subfamily of the BGA glycosidase gene family. The presence of an endoplasmic reticulum retention signal at the carboxy terminus indicates the likely cellular location of PSR3.2. The possible metabolic and regulatory roles of this enzyme during the P_i-starvation response are discussed.     

Triplet nucleotide removal at random positions in a target gene: the tolerance of TEM-1 β-lactamase to an amino acid deletion

The deletion of amino acids is one of the evolutionary mechanisms by which nature adapts the function of proteins. A simple method has been developed that mimics this event in vitro by introducing a deletion of exactly three nucleotides at random positions in a target gene. The method involved the engineering of the mini-Mu transposon to introduce a recognition sequence for the restriction enzyme MlyI. The new transposon, MuDel, was capable of efficient insertion into a target DNA sequence. To determine the efficacy of the method, the bla gene that encodes the TEM-1 β-lactamase was used as the target and a small library containing 22 different sequence variants was created. Of these 22 variants, 8 were identified that conferred resistance to ampicillin on Escherichia coli. Each of the TEM-1 variants possessed a distinct ampicillin minimum inhibitory concentration, ranging from 500 to >10000 μg/ml. Sequence analysis revealed that active TEM-1 variants contained deletions not just in loops but also helices, and included regions known to be involved in catalysis, antibiotic resistance and inhibitor binding. This new technology is transferable to most genes, permitting an extensive analysis of deletion mutations on protein function.