Determination of hemin-binding characteristics of proteins by a combinatorial peptide library approach

Studies of the binding of heme/hemin to proteins or peptides have recently intensified as it became evident that heme serves not only as a prosthetic group, but also as a regulator and effector molecule interacting with transmembrane and cytoplasmic proteins. The iron‐ion‐containing heme group can associate with these proteins in different ways, with the amino acids Cys, His, and Tyr allowing individual modes of binding. Strong coordinate‐covalent binding, such as in cytochrome c, is known, and reversible attachment is also discussed. Ligands for both types of binding have been reported independently, though sometimes with different affinities for similar sequences. We applied a combinatorial approach using the library (X)₄(C/H/Y)(X)₄ to characterize peptide ligands with considerable hemin binding capacities. Some of the library‐selected peptides were comparable in terms of hemin association independently of whether or not a cysteine residue was present in the sequence. Indeed, a preference for His‐based (≈39 %) and Tyr‐based (≈40 %) sequences over Cys‐based ones (≈21 %) was detected. The binding affinities for the library‐selected peptides, as determined by UV/Vis spectroscopy, were in the nanomolar range. Moreover, selected representatives efficiently competed for hemin binding with the human BK channel hSlo1, which is known to be regulated by heme through binding to its heme‐binding domain.     

Oxorhenium-mediated assembly of noncyclic selective integrin antagonists: a combinatorial approach

The parallel oxorhenium-mediated assembly of 288 noncyclic RGD analogues is reported. All complexes contain a NS(2) +S chelating motif that enables the unambiguous coordination of the oxorhenium and oxotechnetium cores. In this study, "modules S" contain a variety of pending guanidinium groups whereas the "NS(2) modules" are made of a series of N-acylated amino acids. Combination of sets of "NS(2) " and "S modules" together with tetrabutylammonium tetrachlorooxorhenate gave the corresponding oxorhenium complexes in good yields and satisfactory purities. Evaluation of these metalloconstructs towards integrins α(V) β(3) , α(IIb) β(3) , and α(V) β(5) led to the identification of micromolar and submicromolar antagonists of theses integrins. These compounds exhibit interesting selectivities and promise attractive applications for the molecular imaging of integrin-dependent pathologies.     

Kinetic data and substrate specificity of a keratinase from Chryseobacterium sp. strain kr6

BACKGROUND: Keratinases are important enzymes for biotechnological processes involving keratin hydrolysis. In this work substrate specificity and kinetic properties of a keratinase from Chryseobaterium sp. were investigated. RESULTS: The optimal conditions for activity of purified keratinase with respect to pH, temperature and sodium chloride concentration were established using factorial design and surface response techniques. The optimum conditions for keratinase activity were pH from 7.4 to 9.2, temperature from 35 °C to 50 °C and NaCl concentration from 50 to 340 mmol L^?1, having azocasein as substrate. Subsequently, the kinetic parameters for this substrate were determined to be K_m = 0.75 mg mL^?1 and V_max = 59.5 U min^?1. The K_i value for 1,10‐phenanthroline was estimated at 0.78 mmol L^?1. The enzyme specificity was evaluated over different synthetic and insoluble substrates. The protease exhibited specificity with selectivity for hydrophobic and positively charged residues. In relation to the insoluble substrates, the enzyme hydrolyzed preferably chicken nails. CONCLUSIONS: This enzyme effectively hydrolyzes insoluble keratin substrates. The knowledge of keratinase properties is an essential step in the development of biotechnological processes involving keratin hydrolysis. Copyright © 2008 Society of Chemical Industry     

Computational studies on bergaptol O-methyltransferase from Ammi majus L.: The substrate specificity

In order to understand the mechanisms of substrate specificity and the interaction between bergaptol and bergaptol O-methyltransferase (BMT), a 3D model of BMT is generated based on the crystal structure of caffeic acid 3-O-methyltransferase (COMT EC 2.1.1.68, PDB code 1KYZ) by using the InsightII/Homology module. With the aid of the molecular mechanics and molecular dynamics methods, the final refined model is obtained and its reliability is further assessed by PROCHECK and ProSa2003. With this model, a flexible docking study is performed and the results indicate that BMT has narrow substrate specificity. Although the homology between both proteins is higher than 65% and all amino acids surrounding the binding site, except four residues, are similar in their sequences, the two proteins exhibit different substrate preferences. The differences in substrate specificity can be explained on the basis of the structures of the protein and the substrate. Our results indicate that His259 may be the catalytic base for the reaction, and Glu320, Glu287 bracket the catalytic His259. Especially, Glu320 forms a weak hydrogen bond with His259 and promotes transfer of an H ion.     

Discovery of a small peptide from combinatorial libraries that can activate the plant immune system by a jasmonic acid signaling pathway

Plants defend themselves by using an innate immune system that is activated in response to a variety of molecules derived from pathogens. These molecules have provided profound insights into the mechanisms of pathogen recognition and subsequent signaling pathways in plants. In the present study, we screened a combinatorial random hexapeptide library for peptides that activate the plant immune system, by using a cell-based high-throughput screening system in which H(2)O(2) generation was monitored. We discovered a novel small peptide (YGIHTH-amide, PIP-1) that triggered H(2)O(2) production in tobacco and tomato cells, but not in Arabidopsis cells. PIP-1 induced significant levels of phytoalexin biosynthesis and defense-related gene expression in tobacco cells; this is likely to be activated by a jasmonic acid pathway.     

Ketoreduction in mycolactone biosynthesis: insight into substrate specificity and stereocontrol from studies of discrete ketoreductase domains in vitro

Mycolactone, a polyketide toxin responsible for the extensive tissue destruction seen in Buruli ulcer, is assembled on a modular polyketide synthase (PKS). Despite operating on structurally different intermediates during synthesis, many of the ketoreductase (KR) domains of the mycolactone (MLS) PKS have identical sequences. This suggests that these enzymes might exhibit an unusually high level of substrate promiscuity. However, we show here that when recombinant mycolactone KR domains are tested with a range of surrogate substrates, their specificity closely matches that of KR domains derived from other PKS systems. In addition, our findings reinforce the role of substrate tethering for achieving stereochemical control in modular PKSs by affecting the delicate energetics of ketoreduction.     

Multicomponent Au/MgO catalysts designed for selective oxidation of carbon monoxide: Application of a combinatorial approach

A complex combinatorial approach has been applied for the design of multicomponent Au/MgO catalysts for CO oxidation in the presence of hydrogen. The combinatorial library design led to unique earlier unknown catalyst compositions. The best catalysts contained significant amount of Pb and Sm, which had never been reported before as useful components of PROX catalysts. In order to increase the diversity of experimental space different pretreatment conditions have been applied prior to catalytic tests. Actually, two catalyst libraries have been tested after (i) a reductive, and (ii) a combined reductive pretreatment. Significantly different optimum compositions have been obtained upon using these two pretreatment procedures. The combined reductive treatment resulted in a more cost effective optimum composition with significantly less components and smaller gold content in comparison to the best catalyst obtained upon using a simple reductive treatment. Further studies showed different ways of promoting action of modifiers. After reductive pretreatment the modifiers suppress the hydrogen consumption, while after combined reductive pretreatment the modifiers lead to the promotion of both oxidation reactions.     

Engineering a chimeric pyrroloquinoline quinone glucose dehydrogenase: improvement of EDTA tolerance, thermal stability and substrate specificity

An engineered Escherichia coli PQQ glucose dehydrogenase (PQQGDH)with improved enzymatic characteristics was constructed by substitutingand combining the gene-encoding protein regions responsiblefor EDTA tolerance, thermal stability and substrate specificity.The protein region responsible for complete EDTA tolerance inAcinetobacter calcoaceticus, which is recognized as the indicatorof high stability in co-factor binding, was elucidated. Theregion is located between 32 and 59% from the N-terminus ofA.calcoaceticus PQQGDH(A27 region) and also corressponds tothe same position from 32 to 59% from the N-terminus in E.coliPQQGDH, though E.coli PQQGDH is EDTA sensitive. We previouslyreported that the C-terminal 3% region of A.calcoaceticus (A3region) played an important role in the increase of thermalstability, and that His775Asn substitution in E.coli PQQGDHresulted in an increase in the substrate specificity of E.coliPQQGDH towards glucose. Based on these findings, chimeric and/ormutated PQQGDHs, E97A3 H775N, E32A27E41 H782N, E32A27E38A3 andE32A27E38A3 H782N were constructed to investigate the compatibilityof two protein regions and one amino acid substitution. His775substitution to Asn corresponded to His782 substitution to Asn(H782N) in chimeric enzymes harbouring the A27 region. Sinceall the chimeric PQQGDHs harbouring the A27 region were EDTAtolerant, the A27 region was found to be compatible with theother region and substituted amino acid responsible for theimprovement of enzymatic properties. The contribution of theA3 region to thermal stability complemented the decrease inthe thermal stability due to the His775 or His782 substitutionto Asn. E32A27E38A3 H782N, which harbours all the above mentionedthree regions, showed improved EDTA tolerance, thermal stabilityand substrate specificity. These results suggested a strategyfor the construction of a semi-artificial enzyme by substitutingand combining the gene-encoding protein regions responsiblefor the improvement of enzyme characteristics. The characteristicsof constructed chimeric PQQGDH are discussed based on the predictedmodel, ß-propeller structure.     

Mechanism and substrate specificity of tRNA-guanine transglycosylases (TGTs): tRNA-modifying enzymes from the three different kingdoms of life share a common catalytic mechanism

Transfer RNA-guanine transglycosylases (TGTs) are evolutionarily ancient enzymes, present in all kingdoms of life, catalyzing guanine exchange within their cognate tRNAs by modified 7-deazaguanine bases. Although distinct bases are incorporated into tRNA at different positions in a kingdom-specific manner, the catalytic subunits of TGTs are structurally well conserved. This review provides insight into the sequential steps along the reaction pathway, substrate specificity, and conformational adaptions of the binding pockets by comparison of TGT crystal structures in complex with RNA substrates of a eubacterial and an archaebacterial species. Substrate-binding modes indicate an evolutionarily conserved base-exchange mechanism with a conserved aspartate serving as a nucleophile through covalent binding to C1' of the guanosine ribose moiety in an intermediate state. A second conserved aspartate seems to control the spatial rearrangement of the ribose ring along the reaction pathway and supposedly operates as a general acid/base. Water molecules inside the binding pocket accommodating interaction sites subsequently occupied by polar atoms of substrates help to elucidate substrate-recognition and substrate-specificity features. This emphasizes the role of water molecules as general probes to map binding-site properties for structure-based drug design. Additionally, substrate-bound crystal structures allow the extraction of valuable information about the classification of the TGT superfamily into a subdivision of presumably homologous superfamilies adopting the triose-phosphate isomerase type barrel fold with a standard phosphate-binding motif.     

Interaction between a Fab fragment against gp41 of human immunodeficiency virus 1 and its peptide epitope: characterization using a peptide epitope library and molecular modeling

The molecular interaction of the Fab fragment of the human monoclonalantibody 3D6, directed against the transmembrane protein gp41of human immunodeficiency virus (HTV) 1, with its peptide epitopeis characterized by a panel of overlapping peptides, a peptideepitope library and molecular modeling techniques. The sequenceCSGKLICTTAVPW, corresponding to amino acids 605–617 ofgp41, was identified as the best binding peptide (K_D = 1x10^-8mol/1). This peptide served as a starting point to prepare acellulose-bound peptide epitope library in which each residueof the epitope is substituted by all L- and D-amino acids, resultingin 494 epitope peptide variants which were subsequently analyzedfor binding 3D6. The library was synthesized to identify residuescritical for binding and to obtain information about the molecularenvironment of the epitope peptide bound to 3D6. Both cysteineresidues, as well as isoleucine 6, threonine 8 and proline 12,of the epitope were highly sensitive to substitution. Usingthe data obtained from the epitope characterization, as wellas a low-resolution electron density map of a 3D6 Fab-peptidecomplex, a 3-D model of the Fab-peptide complex was generatedby molecular modeling. The modeling experiments predict bindingof the peptide, which is cyclized via the two cysteine residues,to a pocket formed dominantly by the hypervariable loops complementaritydetermining regions CDR3L, CDR2H and CDR3H.     

The role of the Cys191-Cys220 disulfide bond in trypsin: new targets for engineering substrate specificity

The S1 binding site of trypsin is cross-linked by the conserved Cys191- Cys220 disulfide bond. The substitution of Cys191 and Cys220 with Ala decreases the activity of trypsin by 20-200-fold as measured by kcat/K(m) for the hydrolysis of amide substrates; in contrast, ester hydrolysis is decreased by < 10-fold. Similar decreases are observed in the hydrolysis of oligopeptide and single amino acid substrates. This decrease in activity results from a decrease in the acylation rate. The substrate binding and deacylation rate are not affected by the loss of the disulfide bond. C191A/C220A binds BPTI with the same affinity as trypsin, although the affinity of benzamidine is decreased 10-fold and the affinity of leupeptin is decreased 1000-fold. The CD spectrum of C191A/C220A displays significant differences from that of trypsin; these differences most likely result from the loss of the disulfide chromophore, although perturbation of enzyme structure cannot be discounted. The loss of the Cys191-Cys220 disulfide has no effect on the stability of trypsin as measured by urea denaturation. Single and double substitutions of Ser at positions 191 and 220 have a similar activity to C191A/C220A. These results indicate that the Cys191-Cys220 disulfide bond is not essential for the function, structure or stability of trypsin.      

The SBASE domain library: a collection of annotated protein segments

SBASE is a database of annotated protein domain sequences representingvarious structural, functional, ligand binding and topogenicsegments of proteins. The current release of SBASE contains27 211 entries which are provided with standardized names inorder to facilitate retrieval. SBASE is cross-referenced tothe major protein and nucleic acid databanks as well as to thePROSITE catalog of protein sequence patterns [Bairoch, A. (1992)Nucleic Acids Res., 20, Suppl., 2013–2118]. SBASE canbe used to establish domain homologies through database searchusing programs such as FASTA [Lipman and Pearson (1985) Science,227, 1436–1441], FASTDB [Brutlag et al. (1990) Comp. Appl.Biosci., 6, 237–245] or BLAST3 [Altschul and Lipman (1990)Proc. Natl. Acad. Sci. USA, 87, 5509–5513], which is especiallyuseful in the case of loosely defined domain types for whichefficient consensus patterns cannot be established. The useof SBASE is illustrated on the DNA binding protein Brain-4.The database and a set of search and retrieval tools are freelyavailable on request to the authors or by anonymous ‘ftp’file transfer from <ftp.icgeb.trieste.it>.     

A hybrid of bovine pancreatic ribonuclease and human angiogenin: an external loop as a module controlling substrate specificity?

A comparison of the sequences of three homologous ribonucleases(RNase A, angiogenin and bovine seminal RNase) identifies threesurface loops that are highly variable between the three proteins.Two hypotheses were contrasted: (i) that this variation mightbe responsible for the different catalytic activities of thethree proteins; and (ii) that this variation is simply an exampleof surface loops undergoing rapid neutral divergence in sequence.Three hybrids of angiogenin and bovine pancreatic ribonuclease(RNase) A were prepared where regions in these loops taken fromangiogenin were inserted into RNase A. Two of the three hybridshad unremarkable catalytic properties. However, the RNase Amutant containing residues 63–74 of angiogenin had greatlydiminished catalytic activity against uridylyl-(3' – 5')-adenosine(UpA), and slightly increased catalytic activity as an inhibitorof translation in vitro. Both catalytic behaviors are characteristicof angiogenin. This is one of the first examples of an engineeredexternal loop in a protein. Further, these results are complementaryto those recently obtained from the complementary experiment,where residues 59–70 of RNase were inserted into angiogenin[Harper and Vallee (1989) Biochemistry, 28, 1875–1884].Thus, the external loop in residues 63–74 of RNase A appearsto behave, at least in part, as an interchangeable ‘module’that influences substrate specificity in an enzyme in a waythat is isolated from the influences of other regions in theprotein.     

Characterization and application of a recombinant dopa decarboxylase from Harmonia axyridis for the efficient biosynthesis of dopamine

Here,a dopa decarboxylase (DDC) from Harmonia axyridis was heterogeneously expressed in Escherichia coli for the efficient biosynthesis of dopamine.For the production of recombinant DDC,the cultivation conditions including IPTG concentration,temperature and induction time were optimized and obtained an optimal specific enzyme activity of 51.72 U·mg-1 crude extracts.After the purification of DDC with a recovery yield of 68.79％,its activity was further characterized.The Vmax,Km,Kcat,and Kcat/Km of DDC for dihydroxyphenylalanine (dopa) were 0.02 mmol·ml-1·s-1,2.328 mmol·ml-1,10435.90 s-1 and 4482.77 ml·mmol-1·s-1,respectively.The highest DDC activity was observed at the condition of pH 7.5 and 45 ℃.With the purified DDC,the feasibility to produce dopamine from L-dopa was evaluated.The optimal yield was determined at the following bioconversion conditions:pH of 7.0,the reaction temper-ature of 40 ℃,0.4 mmol·L-1 of PLP and 4 g·L-1 of L-dopa.Subsequently,a fed-batch process for the pro-duction of dopamine was developed and the effect of oxygen was evaluated.The titer,yield and productivity of dopamine reached up to 21.99 g·L-1,80.88％ and 14.66 g·L-1h-1 at 90 min under anaer-obic condition.     

The structural basis for the altered substrate specificity of the R292D active site mutant of aspartate aminotransferase from E.coli

Two refined crystal structures of aspartate aminotransferasefrom E.coli are reported. The wild type enzyme is in the pyridoxalphosphate (PLP) form and its structure has been determined to2.4 Å resolution, refined to an R-factor of 23.2%. Thestructure of the Arg292Asp mutant has been determined at 2.8Å resolution, refined to an R–factor of 20.3%. Thewild type and mutant crystals are isomorphous and the two structuresare very similar, with only minor changes in positions of importantactive site residues. As residue Arg292 is primarily responsiblefor the substrate charge specificity in the wild type enzyme,the mutant containing a charge reversal at this position mightbe expected to catalyze transamination of arginine as efficientlyas the wild type enzyme effects transamination of aspartate[Cronin,C.N. and Kirsch,J.F. (1988) Biochemistry, 27, 4572–4579].This mutant does in fact prefer arginine over aspartate as asubstrate, however, the rate of catalysis is much slower thanthat of the wild type enzyme with its physiological substrate,aspartate. A comparison of these two structures indicates thatthe poorer catalytic efficiency of R292D, when presented witharginine, is not due to a gross conformational difference, butis rather a consequence of both small side chain and main chainreorientations and the pre–existing active site polarenvironment, which greatly favors the wild type ion pair interaction.