Effect of Feeding Bengal Gram (Cicer arietinum) at Different Protein Levels on Plasma Lipids of Rats

L-Amino acid ligase catalyzes the formation of an α-peptide bond from unprotected L-amino acids in an ATP-dependent manner, and this enzyme is very useful in efficient peptide production. We performed enzyme purification to obtain a novel L-amino acid ligase from Bacillus subtilis NBRC3134, a microorganism producing peptide-antibiotic rhizocticin. Rhizocticins are dipeptide or tripeptide antibiotics and commonly possess L-arginyl-L-2-amino-5-phosphono-3-cis-pentenoic acid. The purification was carried out by detecting L-arginine hydroxamate synthesis activity, and a target enzyme was finally purified 1,280-fold with 0.8% yield. The corresponding gene was then cloned and designated rizA. rizA was 1,242 bp and coded for 413 amino acid residues. Recombinant RizA was prepared, and it was found that the recombinant RizA synthesized dipeptides whose N-terminus was L-arginine in an ATP-dependent manner. RizA had strict substrate specificity toward L-arginine as the N-terminal substrate; on the other hand, the substrate specificity at the C-terminus was relaxed.     

Cluster analysis identifies aminoacid compositional features that indicate Toxoplasma gondii adhesin proteins

Toxoplasma gondii invade host cells using a multi-step process that depends on the regulated secretion of adhesions. To identify key primary sequence features of adhesins in this parasite, we analyze the relative frequency of individual amino acids, their dipeptide frequencies, and the polarity, polarizability and Van der Waals volume of the individual amino acids by using cluster analysis. This method identified cysteine as a key amino acid in the Toxoplasma adhesin group. The best vector algorithm of non-concatenated features was for 2 attributes: the single amino acid relative frequency and the dipeptide frequency. Polarity, polarizability and Van der Waals volume were not good classificatory attributes. Single amino acid attributes clustered unambiguously 67 apicomplexan hypothetical adhesins. This algorithm was also useful for clustering hypothetical Toxoplasma target host receptors. All of the cluster performances had over 70% sensitivity and 80% specificity. Compositional aminoacid data can be useful for improving machine learning-based prediction software when homology and structural data are not sufficient.     

Synthesis and application of dipeptides; current status and perspectives

The functions and applications of l-α-dipeptides (dipeptides) have been poorly studied compared with proteins or amino acids. Only a few dipeptides, such as aspartame (l-aspartyl-l-phenylalanine methyl ester) and l-alanyl-l-glutamine (Ala-Gln), are commercially used. This can be attributed to the lack of an efficient process for dipeptide production though various chemical or chemoenzymatic method have been reported. Recently, however, novel methods have arisen for dipeptide synthesis including a nonribosomal peptide-synthetase-based method and an l-amino acid α-ligase-based method, both of which enable dipeptides to be produced through fermentative processes. Since it has been revealed that some dipeptides have unique physiological functions, the progress in production methods will undoubtedly accelerate the applications of dipeptides in many fields. In this review, the functions and applications of dipeptides, mainly in commercial use, and methods for dipeptide production including already proven processes as well as newly developed ones are summarized. As aspartame and Ala-Gln are produced using different industrial processes, the manufacturing processes of these two dipeptides are compared to clarify the characteristics of each procedure.     

Bacillus subtilis aminopeptidase: Specificity toward amino acid amides,dipeptides, and oligopeptides

Bacillus subtilis aminopeptidase hydrolyzed amino acid amides with a specificity similar to that determined using amino acyl-β-naphthylamides, but at much greater catalytic rates. Neutral and basic amino acid amides were the best substrates. A series of Leu and Lys NH₂-terminal dipeptides hydrolyzed by Co²⁺-activated aminopeptidase showed that the $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ ratios for the Lys substrates were fourfold greater than the corresponding Leu substrates and that catalytic differences reflected the identity of COOH terminal residues. Greatest catalytic rates were obtained when aromatic residues were in the COOH terminal position of the substrate (Trp, Tyr, Phe); but, significant hydrolysis was achieved when aliphatic residues were COOH-terminal in the dipeptide. The Co²⁺-activated enzyme would not hydrolyze peptide bonds composed of the imide nitrogen of Pro, thus, bradykinin was not a substrate. However, the Co²⁺-activated enzyme removed sequentially the first four residues from eledoisin-related peptide and the A chain of bovine insulin.     

Rat intestinal brush border membrane dipeptidyl-aminopeptidase IV: kinetic properties and substrate specificities of the purified enzyme

The kinetic properties and substrate specificities of dipeptidyl-aminopeptidase IV (EC 3.4.14.—) detergent-solubilized and purified from the brush border membrane of rat small intestinal mucosal cells were investigated. Kinetic analysis of purified dipeptidyl-aminopeptidase IV was carried out with a variety of oligopeptides and β-napthylamide derivatives as substrates. In general, peptides with proline penultimate to the amino terminus (XPro, X= amino acid) are more favored substrates while those with alanine (XAla) are hydrolyzed at a slower rate. There is some activity toward substrates having leucine at both the penultimate position and amino terminus (LeuLeu). The activity of the purified enzyme toward GlylProβ-napthylamide derivative is maximal at pH 8.4 in Tris-HCl buffer, with an activation energy of 7.98 kcal/mol. There is no requirement for metal ion. The ability of various dipeptides to inhibit Gly-l-Pro-β-napthylamide derivative hydrolysis was used to determine the binding specificity of the enzyme for the amino-terminal amino acid. These data show that a free amino acid group is necessary for enzymatic activity and increased hydrophobicity of the amino acid at the amino terminus enhances binding.     

Specificity of Pyrrolysyl-tRNA Synthetase for Pyrrolysine and Pyrrolysine Analogs

Pyrrolysine, the 22nd amino acid, is encoded by amber (TAG = UAG) codons in certain methanogenic archaea and bacteria. PylS, the pyrrolysyl-tRNA synthetase, ligates pyrrolysine to tRNA^Pyl for amber decoding as pyrrolysine. PylS and tRNA^Pyl have potential utility in making tailored recombinant proteins. Here, we probed interactions necessary for recognition of substrates by archaeal PylS via synthesis of close pyrrolysine analogs and testing their reactivity in amino acid activation assays. Replacement of the methylpyrroline ring of pyrrolysine with cyclopentane indicated that solely hydrophobic interactions with the ring-binding pocket of PylS are sufficient for substrate recognition. However, a 100-fold increase in the specificity constant of PylS was observed with an analog, 2-amino-6-((R)-tetrahydrofuran-2-carboxamido)hexanoic acid (2Thf-lys), in which tetrahydrofuran replaced the pyrrolysine methylpyrroline ring. Other analogs in which the electronegative atom was moved to different positions suggested PylS preference for a hydrogen-bond-accepting group at the imine nitrogen position in pyrrolysine. 2Thf-lys was a preferred substrate over a commonly employed pyrrolysine analog, but the specificity constant for 2Thf-lys was 10-fold lower than for pyrrolysine itself, largely due to the change in K_m. The in vivo activity of the analogs in supporting UAG suppression in Escherichia coli bearing genes for PylS and tRNA^Pyl was similar to in vitro results, with l-pyrrolysine and 2Thf-lys supporting the highest amounts of UAG translation. Increasing concentrations of either PylS substrate resulted in a linear increase in UAG suppression, providing a facile method to assay bioactive pyrrolysine analogs. These results illustrate the relative importance of the H-bonding and hydrophobic interactions in the recognition of the methylpyrroline ring of pyrrolysine and provide a promising new series of easily synthesized pyrrolysine analogs that can serve as scaffolds for the introduction of novel functional groups into recombinant proteins.     

Probing the Putative Active Site of YjdL: An Unusual Proton-Coupled Oligopeptide Transporter from E. coli

YjdL from E. coli is an unusual proton-coupled oligopeptide transporter (POT). Unlike prototypical POTs, dipeptides are preferred over tripeptides, in particular dipeptides with a positively charged C-terminal residue. To further understand this difference in peptide specificity, the sequences of YjdL and YdgR, a prototypical E. coli POT, were compared in light of the crystal structure of a POT from Shewanella oneidensis. Several residues found in the putative active site were mutated and the activities of the mutated variants were assessed in terms of substrate uptake assays, and changes in specificity in terms of uptake inhibition. Most strikingly, changing the YjdL specific Asp392 to the conserved Ser in YjdL obliterated the preference for a positively charged C-terminal residue. Based on this unique finding and previously published results indicating that the dipeptide N-terminus may interact with Glu388, a preliminary orientation model of a dipeptide in the YjdL cavity is presented. Single site mutations of particularly Ala281 and Trp278 support the presented orientation. A dipeptide bound in the cavity of YjdL appears to be oriented such that the N-terminal side chain protrudes into a sub pocket that opens towards the extracellular space. The C-terminal side chain faces in the opposite direction into a sub pocket that faces the cytoplasm. These data indicated a stabilizing effect on a bulky N-terminal residue by an Ala281Phe variant and on the dipeptide backbone by Trp278. In the presented orientation model, Tyr25 and Tyr58 both appear to be in proximity of the dipeptide backbone while Lys117 appears to be in proximity of the peptide C-terminus. Mutational studies of these conserved residues highlight their functional importance.     

Molecular Cloning and Nucleotide Sequence of the Gene for an Alkaline Protease from Bacillus circulans MTCC 7906

Bacillus circulans MTCC 7906, an extracellular alkaline protease producer was genetically characterized. B. circulans genomic DNA was isolated, oligonucleotide primers specific to alkaline protease gene of B. circulans were designed and its PCR amplification was done. The purified PCR product and pTrcHisA vector were subjected to restriction digestion with NcoI and HindIII and transformed into Escherichia coli DH5-α competent cells. The recombinant expression of alkaline protease gene studied by inducible expression and analysis by SDS-PAGE, established that the alkaline protease protein had an estimated molecular size of 46 kDa. Gene sequencing of the insert from selected recombinant clone showed it to be a 1329 bp gene encoding a protein of 442 amino acids. The sequence was blasted and aligned with known alkaline protease genes for comparison with their nucleotide and amino acid sequences. This identified major matches with three closely related subsp. of B. subtilis (B. subtilis subsp. subtilis strain 168, B. subtilis BSn5 and B. subtilis subsp. spizizenii strain W23). The insert also showed a number of substitutions (mutations) with other sp. of Bacillus which established that alkaline protease of B. circulans MTCC 7906 is a novel gene. The phylogenetic analysis of alkaline protease gene and its predicted amino acid sequences also validated that alkaline protease gene is a novel gene and the same has been accessioned in GenBank with accession number {"type":"entrez-nucleotide","attrs":{"text":"JN645176.1","term_id":"353260575","term_text":"JN645176.1"}}JN645176.1.     

Overexpression and biochemical characterization of beta-1,3-N-acetylgalactosaminyltransferase LgtD from Haemophilus influenzae strain Rd

The lipopolysaccharide of capsule deficient Haemophilus influenzae strain Rd contains an N-acetylgalactosamine residue attached to the terminal globotriose moiety in the Hex5 glycoform. Genome analysis identified an open reading frame HI1578, referred to as lgtD, whose amino acid sequence shows significant level of similarity to a number of bacterial glycosyltransferases involved in lipopolysaccharide biosynthesis. To investigate its function, overexpression and biochemical characterization were performed. Most of the protein was obtained in a highly soluble and active form. By using standard glycosyltransferase assay and HPLC, we show that LgtD is an N-acetylgalactosaminyltransferase with high donor substrate specificity and globotriose is a highly preferred acceptor substrate for the enzyme. The K_m for UDP-GalNAc and globotriose are 58 μM and 8.6 mM, respectively. The amino acid sequence of the enzyme shows the conserved features of family II glycosyltransferases. This is the first N-acetylgalactosaminyltransferase identified from H. influenzae, which shows potential application in large-scale synthesis of globo-series oligosaccharides.     

Bacillusd-stereospecific metallo-amidohydrolase: Active-site metal-ion substitution changes substrate specificity

From investigation of 2000 soil isolates, we identified a d-stereospecific metallo-amidohydrolase that can hydrolyze d-aminoacyl derivatives from the culture supernatant of Bacillus sp. 62E11: 62E11DppA. The enzyme binds two equivalents of zinc, exhibits 70% identity with that of d-aminopeptidases from Bacillus subtilis (DppA). In fact, 62E11DppA has strict specificity toward d-aminoacyl derivatives, i.e., the enzyme shows high activity toward d-aminoacyl benzyl esters and little activity toward d-amino acid containing peptides. Moreover, 62E11DppA exhibits a dramatic change in its activity and substrate specificity by substitution of metal ions in its active site. Based on results of kinetic studies using apo-62E11DppA with various metal ion and substrate concentrations, we propose a possible mechanism for the change in its activity and specificity by substitution of metal ions: the substitution of metal ions in 62E11DppA dramatically changes its activity by altering the substrate specificity.     

Some properties of a homocarnosine-carnosine synthetase isolated from rat brain

—An enzyme from rat brain catalysing the synthesis of the histidine-containing dipeptides carnosine and homocarnosine (l .-histidine: β-alanine ligase (AMP) [EC 6.3.2.11]) was purified about 30-40-fold from a 100,000 g supernatant. Assays were conducted by measuring the incorporation of L-[14C]histidine into carnosine and homocarnosine isolated by paper electrophoresis from the incubation mixture. The ratios of specific activities for the formation of carnosine and homocarnosine were not significantly different for the various purification steps. This was taken as evidence of one enzyme synthesizing both dipeptides. In studying the properties of this enzyme, a pH optimum of 7.4 was shown for carnosine synthesis. The concentrations of amino acid substrates giving maximal synthesis of both dipeptides were in the physiological range found for rat brain. An apparent requirement for ATP, Mg²⁺, and DPN was seen for dipeptide synthesis. A substrate dependent, enzymecatalysed ³²PPi-ATP exchange reaction was observed, suggesting the formation of an aminoacyl-AMP intermediate. Certain other nucleoside triphosphates could substitute for the ATP; this effect showed a specificity toward the dipeptide being synthesized. The apparent requirement for DPN was quite specific, with a number of related compounds having no effect. The stoichiometry of enzyme-catalysed carnosine synthesis was studied. A one to one relationship between carnosine formed and ATP hydrolysed was demonstrated. However, the ratio between carnosine synthesized and DPN hydrolysed was about 6 to 1, indicating a catalytic role for the DPN. The breakdown of DPN did not occur with enzyme alone but was dependent on the presence of substrate.     

Engineering of an Orthogonal Aminoacyl-tRNA Synthetase for Efficient Incorporation of the Non-natural Amino Acid O-Methyl-L-tyrosine using Fluorescence-based Bacterial Cell Sorting

We describe a strategy for the rapid selection of mutant aminoacyl-tRNA synthetases (aaRS) with specificity for a novel amino acid based on fluorescence-activated cell sorting of transformed Escherichia coli using as reporter the enhanced green fluorescent protein (eGFP) whose gene carries an amber stop codon (TAG) at a permissive site upstream of the fluorophore. To this end, a one-plasmid expression system was developed encoding an inducible modified Methanocaldococcus jannaschii (Mj) tyrosyl-tRNA synthetase, the orthogonal cognate suppressor tRNA, and eGFP_UAG in an individually regulatable fashion. Using this system a previously described aaRS with specificity for O-methyl-L-tyrosine (MeTyr) was engineered for 10-fold improved incorporation of the foreign amino acid by selection from a mutant library, prepared by error-prone as well as focused random mutagenesis, for MeTyr-dependent eGFP fluorescence. Applying alternating cycles of positive and negative fluorescence-activated bacterial cell sorting in the presence or in the absence, respectively, of the foreign amino acid was crucial to select for high specificity of MeTyr incorporation. The optimized synthetase was used for the preparative expression of a modified uvGFP carrying MeTyr at position 66 as part of its fluorophore. This biosynthetic protein showed quantitative incorporation of the non-natural amino acid, as determined by mass spectrometry, and it revealed a unique emission spectrum due to the altered chemical structure of its fluorophore. Our combined genetic/selection system offers advantages over earlier approaches that relied wholly or in part on antibiotic selection schemes, and it should be generally useful for the engineering and optimization of orthogonal aaRS/tRNA pairs to incorporate non-natural amino acids into recombinant proteins.     

EFFECTS OF DIPEPTIDES CONTAINING THE AMINO ACID,PROLINE ON THE CHEMOTAXIS OF TETRAHYMENA PYRIFORMIS. EVOLUTIONARY CONCLUSIONS ON THE FORMATION OF HORMONE RECEPTORS AND HORMONES

Our investigations demonstrate that proline-containing dipeptides can provoke a chemosensory response from the unicellular Tetrahymena pyriformis The chemotactic effects of the dipeptides have a close relationship with the side chain and the lipophilicity of the amino-terminal amino acid. Comparison of ‘mirror’ variants of proline-containing dipeptides points to the fact that dipeptides with small side chain and non-polar character amino acids (Gly-Pro, Ala-Pro) are preferred on the amino-terminal end. In the case of amino acids with very variable side chains, small (Pro-Gly) and the large side chain and non-polar character amino acids (Pro-Leu, Pro-Phe) on the carboxyl-terminal end can induce significant chemotactic responses. With valine on any terminus the proline-containing dipeptide induced a weak repellent effect.     

Fermentative production of L-alanyl-L-glutamine by a metabolically engineered Escherichia coli strain expressing L-amino acid alpha-ligase

In spite of its clinical and nutritional importance, l-alanyl-l-glutamine (Ala-Gln) has not been widely used due to the absence of an efficient manufacturing method. Here, we present a novel method for the fermentative production of Ala-Gln using an Escherichia coli strain expressing l-amino acid alpha-ligase (Lal), which catalyzes the formation of dipeptides by combining two amino acids in an ATP-dependent manner. Two metabolic manipulations were necessary for the production of Ala-Gln: reduction of dipeptide-degrading activity by combinatorial disruption of the dpp and pep genes and enhancement of the supply of substrate amino acids by deregulation of glutamine biosynthesis and overexpression of heterologous l-alanine dehydrogenase (Ald). Since expression of Lal was found to hamper cell growth, it was controlled using a stationary-phase-specific promoter. The final strain constructed was designated JKYPQ3 (pepA pepB pepD pepN dpp glnE glnB putA) containing pPE167 (lal and ald expressed under the control of the uspA promoter) or pPE177 (lal and ald expressed under the control of the rpoH promoter). Either strain produced more than 100 mM Ala-Gln extracellularly, in fed-batch cultivation on glucose-ammonium salt medium, without added alanine and glutamine. Because of the characteristics of Lal, no longer peptides (such as tripeptides) or dipeptides containing d-amino acids were formed.     

Action on peptides by wheat carboxypeptidase

A kinetic analysis has been performed with purified wheat carboxypeptidase by the use of N-acyl dipeptides, Z-Gly-Pro-Leu-Gly (Z = benzyloxycarbonyl), angiotensin II and bradykinin. The values of k_cat were dramatically influenced by amino acid residues occupying the penultimate position from the carboxyl terminus of substrates. The structure of the substrate did not appreciably affect the K_m values.     

Chemotaxis of Escherichia coli to pyrimidines: a new role for the signal transducer tap

Escherichia coli exhibits chemotactic responses to sugars, amino acids, and dipeptides, and the responses are mediated by methyl-accepting chemotaxis proteins (MCPs). Using capillary assays, we demonstrated that Escherichia coli RP437 is attracted to the pyrimidines thymine and uracil and the response was constitutively expressed under all tested growth conditions. All MCP mutants lacking the MCP Tap protein showed no response to pyrimidines, suggesting that Tap, which is known to mediate dipeptide chemotaxis, is required for pyrimidine chemotaxis. In order to confirm the role of Tap in pyrimidine chemotaxis, we constructed chimeric chemoreceptors (Tapsr and Tsrap), in which the periplasmic and cytoplasmic domains of Tap and Tsr were switched. When Tapsr and Tsrap were individually expressed in an E. coli strain lacking all four native MCPs, Tapsr mediated chemotaxis toward pyrimidines and dipeptides, but Tsrap did not complement the chemotaxis defect. The addition of the C-terminal 19 amino acids from Tsr to the C terminus of Tsrap resulted in a functional chemoreceptor that mediated chemotaxis to serine but not pyrimidines or dipeptides. These results indicate that the periplasmic domain of Tap is responsible for detecting pyrimidines and the Tsr signaling domain confers on Tapsr the ability to mediate efficient chemotaxis. A mutant lacking dipeptide binding protein (DBP) was wild type for pyrimidine taxis, indicating that DBP, which is the primary chemoreceptor for dipeptides, is not responsible for detecting pyrimidines. It is not yet known whether Tap detects pyrimidines directly or via an additional chemoreceptor protein.     

Discrimination based on Gly and Arg/Ser at position 673 between dipeptidyl-peptidase (DPP) 7 and DPP11, widely distributed DPPs in pathogenic and environmental gram-negative bacteria

Porphyromonas gingivalis, an asaccharolytic gram-negative rod-shaped bacterium, expresses the novel Asp/Glu-specific dipeptidyl-peptidase (DPP) 11 (Ohara-Nemoto, Y. et al. (2011) J. Biol. Chem. 286, 38115–38127), which has been categorized as a member of the S46/DPP7 family that is preferential for hydrophobic residues at the P1 position. From that finding, 129 gene products constituting five clusters from the phylum Bacteroidetes have been newly annotated to either DPP7 or DPP11, whereas the remaining 135 members, mainly from the largest phylum Proteobacteria, have yet to be assigned. In this study, the substrate specificities of the five clusters and an unassigned group were determined with recombinant DPPs from typical species, i.e., P. gingivalis, Capnocytophaga gingivalis, Flavobacterium psychrophilum, Bacteroides fragilis, Bacteroides vulgatus, and Shewanella putrefaciens. Consequently, clusters 1, 3, and 5 were found to be DPP7 with rather broad substrate specificity, and clusters 2 and 4 were DPP11. An unassigned S. putrefaciens DPP carrying Ser⁶⁷³ exhibited Asp/Glu-specificity more preferable to Glu, in contrast to the Asp preference of DPP11 with Arg⁶⁷³ from Bacteroidetes species. Mutagenesis experiments revealed that Arg⁶⁷³/Ser⁶⁷³ were indispensable for the Asp/Glu-specificity of DPP11, and that the broad specificity of DPP7 was mediated by Gly⁶⁷³. Taken together with the distribution of the two genes, all 264 members of the S46 family could be attributed to either DPP7 or DPP11 by an amino acid at position 673. A more compelling phylogenic tree based on the conserved C-terminal region suggested two gene duplication events in the phylum Bacteroidetes, one causing the development of DPP7 and DPP11 with altered substrate specificities, and the other producing an additional DPP7 in the genus Bacteroides.     

S1 site residues of Lactococcus lactis prolidase affect substrate specificity and allosteric behaviour

Lactococcus lactis prolidase preferably hydrolyzes Xaa-Pro dipeptides where Xaa is a hydrophobic amino acid. Anionic Glu-Pro and Asp-Pro dipeptides cannot be hydrolyzed at any observable rates and the hydrolysis of cationic Arg-Pro and Lys-Pro dipeptides is at about one tenth of the rate of Leu-Pro. It was hypothesized that the hydrophobic residues in the S₁ site were responsible for this substrate specificity, thus the residues in the S₁ site were substituted with hydrophilic residues. The substitution of Leu193 and Val302 revealed that these residues influenced the substrate specificity. The introduction of a cationic residue, L193R, allowed Asp-Pro to be utilized as a substrate at 37.0% of the rate of Leu-Pro, and the anionic mutation, V302D, yielded mutants that could hydrolyze Asp-Pro, Arg-Pro and Lys-Pro at 25.9 to 57.4% rates. Interestingly, these mutants of S₁ site residues eliminated the allosteric behaviour of L. lactis prolidase that makes this enzyme unique among known prolidases. Results of pH dependency, thermal dependency, and molecular modelling suggested that these observed changes were due to the alteration of the interactions among catalytic zinc cations, Arg293, His296, and the mutated residues.