Galina G. Chestukhina

A new subfamily of microbial serine proteinases? Structural similarities of Bacillus thuringiensis and Thermoactinomyces vulgaris extracellular serine proteinases

Abstract Extracellular serine proteinases produced by two taxonomically remote microorganisms - B. thuringiensis and T. vulgaris were shown to share common structural and functional features. Both enzymes contain cysteine residue apparently essential for their activity. Their N-terminal sequences are clearly homologous (10 coinciding residues among 14 compared), whereas only marginal extent of homology could be found when the N-terminal sequences of these enzymes were aligned with those of subtilisins. It is suggested that within the family of evolutionary related bacterial serine proteinases exists a subfamily of SH-containing serine proteinases.

Bacillus intermedius Glutamyl Endopeptidase. Molecular Cloning and Nucleotide Sequence of the Structural Gene

The glutamyl endopeptidase gene of Bacillus intermedius was cloned from a genomic library expressed in Bacillus subtilis and sequenced (EMBL accession number Y15136). The encoded preproenzyme contains 303 amino acid residues; the mature 23-kDa enzyme consists of 215 residues. The mature enzyme reveals 38% of identical residues when aligned with the glutamyl endopeptidase from Bacillus licheniformis, whereas only five invariant residues were found among all known glutamyl endopeptidases. The amino acid residues that form the catalytic triad (H47, D98, and S171) as well as H186 participating in the binding of the substrate carboxyl group were identified. It seems that the structural elements responsible for the function of glutamyl endopeptidases from various sources are highly variable.

Nucleotide sequence of a novel δ‐endotoxin gene cryIg of Bacillus thuringiensis ssp. galleriae

A gene cryIg coding for entomocidal protein δ‐endotoxin of Bacillus thuringiensis ssp. galleriae str. 11‐67 named CryIG has been cloned and sequenced (EMBL accession number X58120). The deduced amino acid sequence that contains 1156 amino acid residues shows only 28% of identical residues, when compared with other δ‐endotoxins of the CryI family. The extent of identity is substantially higher for some regions of the sequence (‘conserved blocks’), that presumably bear important structural or functional properties. This implies that CryIG δ‐endotoxin follows the same type of polypeptide chain folding as other Cryl proteins, whereas peculiarities of primary structure help to explain its unique specificity.

Interaction of 65- and 62-kD proteins from the apical membranes of the Aedes aegypti larvae midgut epithelium with Cry4B and Cry11A endotoxins of Bacillus thuringiensis

A protein with the molecular weight of 65 kD is the only component of Aedes aegypti larvae BBM capable to specifically bind mosquitocidal toxins Cry4B and Cry11A of Bacillus thuringiensis. This protein lacks the leucine aminopeptidase activity which is characteristic for the toxin-binding proteins from the membranes of caterpillars. Cry-toxins inactive against A. aegypti larvae either fail to bind to the 65-kD protein and to a putative product of its proteolysis with the molecular weight of 62 kD (Cry1Ab), or bind but do not compete for this binding with mosquitocidal proteins (Cry9A). The proteolytic splitting out of the first five α-helices in the Cry4B toxin molecule does not affect its binding to the 65- and 62-kD proteins, but an additional removal of 20-30 amino acids from the C-terminal of the molecule sharply spoils this binding. Monosaccharide residues are not involved in the binding of the 65- and 62-kD proteins with Cry4B, Cry11A, and Cry9A.

Molecular cloning and primary structure of Thermoactinomyces vulgaris carboxypeptidase T A metalloenzyme endowed with dual substrate specificity

A gene coding for an extracellular Zn‐carboxypeptidase of Thermoactinomyces vulgaris has been cloned and sequenced (EMBL X56901). This enzyme named carboxypeptidase T reveals simultaneously both types of substrate specificity characteristic of mammalian carboxypeptidases A and B. The carboxypeptidase T gene is primarily expressed in E. coli as a non‐active preproenzyme with an additional 98 amino acid residues at the N‐terminus. Primary structure alignment or mature carboxypeptidase T and mammalian metallocarboxypeptidases demonstrated 25–30% overall identity but a full preservation of presumed catalytically important residues. These observations imply a basic uniformity of the general catalytic mechanism for enzymes of that class produced by evolutionarily remote organisms.

Reconstruction of Bacillus thuringiensis ssp. israelensis Cry11a endotoxin from fragments corresponding to its N- and c-moieties restores its original biological activity

Subtilisin hydrolyzes Cry11A endotoxin (of 70 kD) produced by Bacillus thuringiensis ssp. israelensis to fragments of 33- and 36-kD, which correspond to N- and C-terminal halves of the endotoxin molecule. Thermitase (a serine protease from Thermoactinomyces vulgaris) and insect gut proteases from Diptera and Lepidoptera exhibit the same hydrolytic effect on Cry11A. Hydrolyzates maintain high toxicity with respect to larvae of Aedes aegypti, Anopheles stephensi, and Culex pipiens. The 33- and 36-kD Cry11A endotoxin components purified by ion-exchange chromatography from the subtilisin hydrolyzate were inactive; however, equimolar mixture of these proteins exhibited almost the same activity as the initial hydrolyzate.

Propeptide of the metalloprotease of Brevibacillus brevis 7882 is a strong inhibitor of the mature enzyme

A metalloprotease gene of Brevibacillus brevis (npr) was expressed in Escherichia coli in a soluble form as native Npr precursor. A significant fraction of the precursor was spontaneously processed, producing the N‐terminal propeptide and the mature enzyme. A strong inhibition of the mature Npr by its own propeptide in the crude lysate was observed even in the absence of the covalent linkage between them. Pure precursor, propeptide and the mature Npr were isolated and kinetic parameters of the mature enzyme inhibition by the propeptide were determined. The inhibition is of the tight‐binding competitive type with K i 0.17 nM. Inhibition of metalloproteases from Brevibacillus megaterium and thermolysine by the heterologous propeptide of the Npr from B. brevis was much weaker or none.

Structural features of crystal-forming proteins produced by Bacillus thuringiensis subspecies israelensis

Bacillus thuringiensis ssp. israelensis δ‐Endotoxin Entomocidal crystal Insecticide Mosquito

Two novel delta-endotoxin gene families cry26 and cry28 from Bacillus thuringiensis ssp. finitimus.

Genes cry26Aa1 and cry28Aa1 were cloned from Bacillus thuringiensis ssp. finitimus strain B‐1166 VKPM. This strain forms insecticidal crystal bodies either outside or inside the exosporium. The deduced amino acid sequence of the cry26Aa1 gene product included seven residues determined to be an N‐terminal part of a chymotrypsin‐treated delta‐endotoxin isolated from the same strain. Earlier this protein was detected in both free and spore‐associated types of crystals [Revina et al., Biokhimia (1999) in press]. Neither BtI nor BtII promoter sequences were found upstream of the open reading frames in both genes. Southern hybridization has shown that the surroundings of both genes at least 3 kb upstream and downstream of the open reading frames are unique. We suggest that the protein Cry26Aa1 in both types of crystal bodies is synthesized under the control of one and the same genomic locus.

Limited proteolysis of Bacillus thuringiensis CryIG and CryIVB delta-endotoxins leads to formation of active fragments that do not coincide with the structural domains.

Bacillus thuringiensis “true” toxins consist of three domains: the N-terminal, α-helical domain followed by two β-structural domains. Their limited proteolysis does not proceed at the domain boundaries, but is directed to the loops within the domains. There are at least two patterns of the limited proteolysis of “true” toxins. The first pattern, observed for CryIA and CryIVD δ-endotoxins, results in the proteolysis of the loops connecting β-strands of the second domain. The second pattern, detected for CryIG and CryIVB proteins, consists in the cleavage of the loop connecting the fifth and the sixth α-helixes of the first domain. The choice between the routes depends on the size, sequence, and dynamics of the loop that define its accessibility to a proteinase. Bioassay of CryIG and CryIVB δ-endotoxin fragments indicates that only two α-helixes, the sixth and the seventh within the first domain, followed by the two β-structural domains are sufficient for the insecticidal activity.