Structure of the carboxypeptidase B complex with N-sulfamoyl-L-phenylalanine – a transition state analog of non-specific substrate

Carboxypeptidase B (EC 3.4.17.2) (CPB) is commonly used in the industrial insulin production and as a template for drug design. However, its ability to discriminate substrates with hydrophobic, hydrophilic, and charged side chains is not well understood. We report structure of CPB complex with a transition state analog N-sulfamoyl-L-phenylalanine solved at 1.74Å. The study provided an insight into structural basis of CPB substrate specificity. Ligand binding is affected by structure-depended conformational changes of Asp255 in S1’-subsite, interactions with Asn144 and Arg145 in C-terminal binding subsite, and Glu270 in the catalytic center. Side chain of the non-specific substrate analog SPhe in comparison with that of specific substrate analog SArg (reported earlier) not only loses favorable electrostatic interactions and two hydrogen bonds with Asp255 and three fixed water molecules, but is forced to be in the unfavorable hydrophilic environment. Thus, Ser207, Gly253, Tyr248, and Asp255 residues play major role in the substrate recognition by S1’-subsite.     

Synthesis of cyclic analogues of loop 4 of nerve growth factor

Cyclic peptides cyclo(-Gly-Asp-Glu-Lys-), cyclo(-Gly-Gly-Asp-Glu-Lys-) and cyclo(-Gly-Gly-Gly-Asp-Glu-Lys-) were synthesized as models of theβ-turn of nerve growth factor loop 4. The corresponding protected linear precursors were obtained in 52–83% yields by the solid-phase method with the use of the Fmoc/Bu ^t strategy and a chlorotrityl anchor group. The cyclization was carried out with benzotriazolyloxytris(dimethylamino)phosphonium (BOP) hexafluorophosphate, N-[(1H-benzotriazole-1-yl)-(dimethylamino)methylene]-N-methylmetanaminium-N-oxide (HBTU) hexafluorophosphate, and diphenylphosphorylazide (DPPA) at a dilution of 10^?3 M. The distribution of reaction products was studied for each cyclopeptide in dependence on the type of the coupling agent. The use of DPPA was shown to completely inhibit the formation of cyclodimers in the synthesis of five-and six-membered cyclopeptides; however, in the case of a four-membered peptide, an additional tenfold dilution of the reaction mixture was necessary to achieve the effect. The identification of several byproducts during the synthesis showed that the elongation of the polypeptide chain using the BOP reagent can be complicated by substantial racemization, and the cleavage of the chlorotrityl anchor group by 0.5% TFA in dichloromethane proceeds with insufficient selectivity and is accompanied by the premature Boc deblocking of the lysine side function.     

ISSR Analysis of Variability of Cultivated Form and Varieties of Pomegranate (<Emphasis Type="Italic">Punica granatum</Emphasis> L.) from Azerbaijan

The article presents the results of a study of genetic polymorphism for the first time carried out on pomegranate varieties and forms of Azerbaijan origin using molecular markers. In total, 102 PCR fragments were identified, of which 80 were polymorphic. The high level of polymorphism (75.5%) and the rich genetic diversity were identified among the studied pomegranate collection. As a result of data analysis and on the basis of the values of the basic parameters (PIC, EMR, MI, RP, MRP) determining informativeness of markers, all 14 ISSR primers were suitable for genotyping pomegranate accessions. The most effective markers (UBC808, UBC811, UBC834, and UBC840) were identified among the set of primers tested. A dendrogram was constructed on the basis of the data obtained, which made it possible to group genotypes into 16 major clusters. The genetic similarity index ranged from 0.032 to 0.94. The study of the genetic relationship of different pomegranate varieties confirms the effectiveness of the ISSR method, which makes it possible to determine the level of genetic diversity, as well as to establish the relationship among the studied pomegranate accessions.     

Extracellular serine proteinase from Bacillus licheniformis

The serine proteinase from B. licheniformis was purified by affinity chromatography on the sorbent obtained by attachment of p-(omega-aminomethyl)-phenylboronic acid via an amino group to CH-Sepharose. The use of this sorbent specific to the serine proteinases active sites resulted in a 35-fold purification of the enzyme with an apparent activity yield of 288%. Such a high activity yield is due to a removal of the enzyme inhibitors. The N-terminal sequence of B. licheniformis extracellular serine proteinase traced for 35 amino acid residues coincides with that of subtilisin Carlberg, a serine proteinase presumed to be secreted by a B. subtilis strain. Since the amino acid composition as well as the functional properties of these two enzymes did not reveal any noticeable differences, it was assumed that both proteinases are very similar, if not identical. This conclusion leads to reconsideration of the existing concept on an extremely fast rate of subtilisin evolution. Three multiple forms of B. licheniformis extracellular serine proteinase were found to differ only in their net charges, presumably as a result of partial deamidation of Asn or Gln residues within their structure.     

Leu254 residue and calcium ions as new structural determinants of carboxypeptidase T substrate specificity

New determinants of Thermoactinomyces vulgaris carboxypeptidase T (CPT) substrate specificity--structural calcium ions and Leu254 residue--were found by means of steady-state kinetics and site-directed mutagenesis. The removal of calcium ions shifted the selectivity profile of hydrolysis of tripeptide substrates with C-terminal Leu, Glu, and Arg from 64/1.7/1 to 162/1.3/1. Substitution of the hydrophobic Leu254 in CPT for polar Asn did not change hydrolysis efficiency of substrates with C-terminal Leu and Arg, but resulted in more than 28-fold decrease in activity towards the substrate with C-terminal Glu. It is shown that the His68 residue is not a structural determinant of CPT specificity.     

New insights into the history of domesticated and wild apricots and its contribution to Plum pox virus resistance

Studying domesticated species and their wild relatives allows understanding of the mechanisms of population divergence and adaptation, and identifying valuable genetic resources. Apricot is an important fruit in the Northern hemisphere, where it is threatened by the Plum pox virus (PPV), causing the sharka disease. The histories of apricot domestication and of its resistance to sharka are however still poorly understood. We used 18 microsatellite markers to genotype a collection of 230 wild trees from Central Asia and 142 cultivated apricots as representatives of the worldwide cultivated apricot germplasm; we also performed experimental PPV inoculation tests. The genetic markers revealed highest levels of diversity in Central Asian and Chinese wild and cultivated apricots, confirming an origin in this region. In cultivated apricots, Chinese accessions were differentiated from more Western accessions, while cultivated apricots were differentiated from wild apricots. An approximate Bayesian approach indicated that apricots likely underwent two independent domestication events, with bottlenecks, from the same wild population. Central Asian native apricots exhibited genetic subdivision and high frequency of resistance to sharka. Altogether, our results contribute to the understanding of the domestication history of cultivated apricot and point to valuable genetic diversity in the extant genetic resources of wild apricots.     

Three-dimensional structure of carboxypeptidase T from Thermoactinomyces vulgaris in complex with N-BOC-L-leucine

The 3D structure of recombinant bacterial carboxypeptidase T (CPT) in complex with N-BOC-L-leucine was determined at 1.38 Å resolution. Crystals for the X-ray study were grown in microgravity using the counter-diffusion technique. N-BOC-L-leucine and SO ₄ ^2? ion bound in the enzyme active site were localized in the electron density map. Location of the leucine side chain in CPT-N-BOC-L-leucine complex allowed identification of the S1 subsite of the enzyme, and its structure was determined. Superposition of the structures of CPT-N-BOC-L-leucine complex and complexes of pancreatic carboxypeptidases A and B with substrate and inhibitors was carried out, and similarity of the S1 sub-sites in these three carboxypeptidases was revealed. It was found that SO ₄ ^2? ion occupies the same position in the S1’ subsite as the C-terminal carboxy group of the substrate.     

Isoelectric focusing of a preparation of proteolytic enzymes from Streptomyces 771

The proteolytic enzymes contained in the preparation from Streptomyces 771 have been separated by isoelectric focusing in the sucrose density gradient at pH 3-10. The following enzymes have been identified: three multiple forms of neutral metal proteinase (pI 5.1, 6.37, 7.8) each of which splits DNP-Gly-Gly decreases-Val-ArgOMe; elastase-like metal proteinase active with respect to RBB-elestin with pI 10.68; metal-dependent peptidases: leucin aminopeptidase active with respect to L-RBB-elastin with pI 10.68 metal-dependent peptidases: leucin aminopeptidase active with respect to L-leucin n-nitroanilide and L-leucin beta-naphtylamide with pI 7.65, 7.15, 6.67, 6.45, 5.7, 5.35, 5.22, 4.83; carboxy peptidase with pI 5.95, 6.37; serine metal-dependent subtilisin-like proteinase active with respect to 2-Ala-Ala-LeupNA, 2-Gly-Gly-LeupNA, 2-Ala-LeupNA; two multiple forms of serine trypsin-like proteinase active with respect to BAEE and BApNA with pI 4.35, 4.76; serine chymotrypsin-like proteinase with pI 8.68 active with respect to ATEE.