In silico and in vivo analysis of signal peptides effect on recombinant glucose oxidase production in nonconventional yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Signal peptide (SP) is an important factor and biobrick in the production and secretion of recombinant proteins. The aim of this study was in silico and in vivo analysis of SPs effect on the production of recombinant glucose oxidase (GOX) in Yarrowia lipolytica. Several in silico softwares, namely SignalP4, Signal-CF, Phobius, WolfPsort 0.2, SOLpro and ProtParam, were used to analyse the potential of 15 endogenous and exogenous SPs for the secretion of recombinant GOX in Y. lipolytica. According to in silico results, the SP of GOX was predicted as suitable in terms of high secretory potential and of protein solubility and stability which is chosen for in vivo analysis. The recombinant Y. lipolytica strain produced 280 U/L of extracellular GOX after 7 days in YPD medium. The results show that the SP of GOX can be applied to efficient production of extracellular heterologous proteins and metabolic engineering in Y. lipolytica.     

Point mutation of a tyrosine in the linker region of Syk results in a gain of function

The protein tyrosine kinase Syk plays an essential role in Fc epsilon RI-mediated histamine release in mast cells by regulating the phosphorylation of other proteins. We investigated the functional role of a putative Syk phosphorylation site, Tyr317. This tyrosine in the linker region of Syk is a possible site for binding by the negative regulator Cbl. Syk with Tyr317 mutated to Phe (Y317F) was expressed in a Syk-negative variant of the RBL-2H3 mast cells. Compared with cells expressing wild-type Syk, expression of the Y317F mutant resulted in an increase in the Fc epsilon RI-mediated tyrosine phosphorylation of phospholipase C-gamma and a dramatic enhancement of histamine release. The in vivo Fc epsilon RI-induced tyrosine phosphorylation of wild-type Syk and that of the Y317F mutant were similar. Although the Fc epsilon RI-induced tyrosine phosphorylation of total cellular proteins was enhanced in the cells expressing the Y317F Syk, the phosphorylation of some other molecules, including the receptor subunits, Vav and mitogen-activated protein kinase, was not increased. The Fc epsilon RI-induced phosphorylation of Cbl was downstream of Syk kinase activity and was unchanged by expression of the Y317F mutation. These data indicate that Tyr317 in the linker region of Syk functions to negatively regulate the signals leading to degranulation.     

Functions of the major tyrosine phosphorylation site of the PDGF receptor beta subunit.

Two tyrosine phosphorylation sites in the human platelet-derived growth factor receptor (PDGFR) beta subunit have been mapped previously to tyrosine (Y)751, in the kinase insert, and Y857, in the kinase domain. Y857 is the major site of tyrosine phosphorylation in PDGF-stimulated cells. To evaluate the importance of these phosphorylations, we have characterized the wild-type (WT) and mutant human PDGF receptor beta subunits in dog kidney epithelial cells. Replacement of either Y751 or Y857 with phenylalanine (F) reduced PDGF-stimulated DNA synthesis to approximately 50% of the WT level. A mutant receptor with both tyrosines mutated was unable to initiate DNA synthesis, as was a kinase-inactive mutant receptor. Transmodulation of the epidermal growth factor receptor required Y857 but not Y751. We also tested the effects of phosphorylation site mutations on PDGF-stimulated receptor kinase activity. PDGF-induced tyrosine phosphorylation of two cellular proteins, phospholipase C gamma 1 (PLC gamma 1) and the GTPase activating protein of Ras (GAP), was assayed in epithelial cells expressing each of the mutant receptors. Tyrosine phosphorylation of GAP and PLC gamma 1 was reduced markedly by the F857 mutation but not significantly by the F751 mutation. Reduced kinase activity of F857 receptors was also evident in vitro. Immunoprecipitated WT receptors showed a two- to fourfold increase in specific kinase activity if immunoprecipitated from PDGF-stimulated cells. The F751 receptors showed a similar increase in activity, but F857 receptors did not. Our data suggest that phosphorylation of Y857 may be important for stimulation of kinase activity of the receptors and for downstream actions such as epidermal growth factor receptor transmodulation and mitogenesis.     

In silico characterization and transcriptomic analysis of nif family genes from <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC7120

In silico approaches in conjunction with morphology, nitrogenase activity, and qRT-PCR explore the impact of selected abiotic stressor such as arsenic, salt, cadmium, copper, and butachlor on nitrogen fixing (nif family) genes of diazotrophic cyanobacterium Anabaena sp. PCC7120. A total of 19 nif genes are present within the Anabaena genome that is involved in the process of nitrogen fixation. Docking studies revealed the interaction between these nif gene-encoded proteins and the selected abiotic stressors which were further validated through decreased heterocyst frequency, fragmentation of filaments, and downregulation of nitrogenase activity under these stresses indicating towards their toxic impact on nitrogen fixation potential of filamentous cyanobacterium Anabaena sp. PCC7120. Another appealing finding of this study is even though having similar binding energy and similar interacting residues between arsenic/salt and copper/cadmium to nif-encoded proteins, arsenic and cadmium are more toxic than salt and copper for nitrogenase activity of Anabaena which is crucial for growth and yield of rice paddy and soil reclamation.     

Characterization of the lamin analogue NMCP2 in the monocot <Emphasis Type="Italic">Allium cepa</Emphasis>

Nuclear lamina organization is similar in metazoan and plants though the latter lack orthologs of lamins, the main components of the metazoan lamina. Current evidence suggests that Nuclear Matrix Constituent Proteins (NMCPs) are the lamin analogues in plants as these proteins share several key features: higher-order secondary structure and domain layout, subnuclear distribution, and involvement in the regulation of nuclear shape and size, as well as in higher-order chromatin organization. Previously, we studied the NMCP family in flowering plants (angiosperms), in which it comprises two phylogenetic groups: NMCP1 and NMCP2. At present, in silico information about NMCP proteins in embryophytes is relatively advanced, though very few proteins, most of them of the NMCP1 type, have been extensively studied in vivo. We previously characterized the NCMP1 protein in the monocot Allium cepa. Here, we report the key features of a second protein of this species NMCP2, which presents a conserved sequence and domain layout. Immunofluorescence and immunoelectronmicroscopy evidence co-localization of endogenous AcNMCP2 and AcNMCP1 in the lamina, while Western blotting and immunoconfocal microscopy reveal a similar pattern of expression and distribution of both NMCP proteins in different root tissues. Our results provide novel insight about endogenous NMCP2-type proteins and complete the characterization of the NMCP family in A. cepa, thus advancing the current understanding of these structural proteins constituting the plant lamina.     

Platelet-derived growth factor (PDGF)-dependent association of phospholipase C-gamma with the PDGF receptor signaling complex.

We investigated the interaction of phospholipase C-gamma (PLC-gamma) with wild-type and mutant forms of the platelet-derived growth factor (PDGF) beta-receptor both in vivo and in vitro. After PDGF treatment of CHO cell lines expressing wild-type or either of two mutant (delta Ki and Y825F) PDGF receptors, PLC-gamma became tyrosine phosphorylated and associated with the receptor proteins. The receptor association and tyrosine phosphorylation of PLC-gamma correlated with the ability of these receptors to mediate ligand-induced phosphatidylinositol turnover. However, both the delta Ki and Y825F mutant receptors were deficient in transmitting mitogenic signals, suggesting that the PDGF-induced tyrosine phosphorylation and receptor association of PLC-gamma are not sufficient to account for the growth-stimulatory activity of PDGF. Wild-type and delta Ki mutant PDGF receptor proteins expressed with recombinant baculovirus vectors also associated in vitro with mammalian PLC-gamma. However, baculovirus-expressed c-fms, v-fms, c-src, and Raf-1 proteins failed to associate with PLC-gamma under similar conditions. Phosphatase treatment of the baculovirus-expressed PDGF receptor greatly decreased its association with PLC-gamma. This requirement for receptor phosphorylation was also observed in vivo, where PLC-gamma could not associate with a mutant PDGF receptor (K602A) defective in autophosphorylation. PLC-gamma also coimmunoprecipitated with two other putative receptor substrates, the serine-threonine kinase Raf-1 and the 85-kilodalton phosphatidylinositol-3' kinase, presumably through its association with the ligand-activated receptor. Furthermore, baculovirus-expressed Raf-1 phosphorylated purified PLC-gamma in vitro at sites which showed increased serine phosphorylation in vivo in response to PDGF. These results suggest that PDGF directly influences PLC activity by inducing the association of PLC-gamma with a receptor signaling complex, resulting in increased tyrosine and serine phosphorylation of PLC-gamma.     

LFchimera protects HeLa cells from invasion by <Emphasis Type="Italic">Yersinia</Emphasis> spp. in vitro

Yersinia pestis is the causative agent of plague. As adequate antibiotic treatment falls short and currently no effective vaccine is available, alternative therapeutic strategies are needed. In order to contribute to solving this problem we investigated the therapeutic potential of the peptide construct LFchimera against the safer-to-handle Y. pestis simulants Yersinia enterocolitica and Yersinia pseudotuberculosis in vitro. LFchimera is a heterodimeric peptide construct mimicking two antimicrobial domains of bovine lactoferrin, i.e. lactoferrampin and lactoferricin. LFchimera has been shown to be a potent antimicrobial peptide against a variety of bacteria in vitro and in vivo. Also Y. enterocolitica and Y. pseudotuberculosis have been shown to be susceptible for LFchimera in vitro. As Yersiniae spp. adhere to and invade host cells upon infection, we here investigated the effects of LFchimera on these processes. It was found that LFchimera has the capacity to inhibit host-cell invasion by Yersiniae spp. in vitro. This effect appeared to be host-cell mediated, not bacteria-mediated. Furthermore it was found that exposure of human HeLa epithelial cells to both LFchimera and the bacterial strains evoked a pro-inflammatory cytokine release from the cells in vitro.     

Cloning and functional identification of farnesyl diphosphate synthase from <Emphasis Type="Italic">Pinus massoniana</Emphasis> Lamb

Farnesyl diphosphate synthase (FPPS) is a key isoprenyl diphosphate synthase (IDS), which provides synthetic precursors to the terpenoid metabolic pathway. We isolated and characterized a Pinus massoniana FPPS (PmFPPS) gene which encodes a putative farnesyl diphosphate synthase from P. massoniana Lamb. In silico domain analysis revealed that PmFPPS contained all five conserved IDS domains and was homologous to FPPSs from other plant species. An in vitro enzymatic activity assay resulted in an optimum pH, temperature, and Mg²⁺ concentration of 7.0–7.5, 25 °C, and 1.2 mM, respectively. To identify the function of PmFPPS in vivo, sense and antisense expression vectors were constructed and transformed into tobacco using a constitutive cauliflower mosaic virus-35S promoter. The overexpression of PmFPPS in transgenic plants had higher squalene contents than the control, and the downregulated transgenic plants had lower squalene contents than the control. These results indicate that PmFPPS performs a regulatory role in triterpene biosynthesis.     

Inhibition of Bcr serine kinase by tyrosine phosphorylation.

The first exon of the BCR gene encodes a new serine/threonine protein kinase. Abnormal fusion of the BCR and ABL genes, resulting from the formation of the Philadelphia chromosome (Ph), is the hallmark of Ph-positive leukemia. We have previously demonstrated that the Bcr protein is tyrosine phosphorylated within first-exon sequences by the Bcr-Abl oncoprotein. Here we report that in addition to tyrose 177 (Y-177), Y-360 and Y283 are phosphorylated in Bcr-Abl proteins in vitro. Moreover, Bcr tyrosine 360 is phosphorylated in vivo within both Bcr-Abl and Bcr. Bcr mutant Y177F had a greatly reduced ability to transphosphorylate casein and histone H1, whereas Bcr mutants Y177F and Y283F had wild-type activities. In contrast, the Y360F mutation had little effect on Bcr's autophosphorylation activity. Tyrosine-phosphorylated Bcr, phosphorylated in vitro by Bcr-Abl, was greatly inhibited in its serine/threonine kinase activity, impairing both auto- and transkinase activities of Bcr. Similarly, the isolation of Bcr from cells expressing Bcr-Abl under conditions that preserve phosphotyrosine residues also reduced Bcr's kinase activity. These results indicate that tyrosine 360 of Bcr is critical for the transphosphorylation activity of Bcr and that in Ph-positive leukemia, Bcr serine/threonine kinase activity is seriously impaired.     

Effect of H<Subscript>2</Subscript>O<Subscript>2</Subscript> on tyrosine phosphorylation of pea proteins

Changes in tyrosine phosphorylation of soluble polypeptides of pea (Pisum sativum L.) roots were revealed under the action of exogenous hydrogen peroxide in situ and in vitro. The polypeptides whose tyrosine phosphorylation in situ was vanadate-sensitive were identified. A thiol agent dithiothreitol and the antioxidant ascorbic acid reversed the effect of hydrogen peroxide in vitro. The results indicate that tyrosine phosphorylation of pea proteins is a subject to redox regulation.     

<Emphasis Type="Italic">Corynebacterium diphtheriae</Emphasis> HmuT: dissecting the roles of conserved residues in heme pocket stabilization

The heme-binding protein HmuT is part of the Corynebacterium diphtheriae heme uptake pathway and is responsible for the delivery of heme to the HmuUV ABC transporter. HmuT binds heme with a conserved His/Tyr heme axial ligation motif. Sequence alignment revealed additional conserved residues of potential importance for heme binding: R237, Y272 and M292. In this study, site-directed mutations at these three positions provided insight into the nature of axial heme binding to the protein and its effect on the thermal stability of the heme-loaded protein fold. UV–visible absorbance, resonance Raman (rR) and thermal unfolding experiments, along with collision-induced dissociation electrospray ionization mass spectrometry, were used to probe the contributions of each mutated residue to the stability of ? HmuT. Thermal unfolding and rR experiments revealed that R237 and M292 are important residues for heme binding. Arginine 237 is a hydrogen-bond donor to the phenol side chain of Y235, which serves as an axial heme ligand. Methionine 292 serves a supporting structural role, favoring the R237 hydrogen-bond donation, which elicits a, heretofore, unobserved modulating influence on π donation by the axial tyrosine ligand in the heme carbonyl complex, HmuT–CO.     

Antimicrobial activity of endogenous peptides of the moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Plant and animal cells contain pools of endogenous peptides, which are the degradation products of functionally active proteins. It is known that these peptides can possess biological activity; however, the functions of most of them are unknown. The goal of the present study was to estimate the antimicrobial potential of endogenous peptides resulting from the degradation of functional proteins in cells of the moss Physcomitrella patens. Earlier, 117 peptides possessing an antimicrobial potential predicted in silico have been identified in the peptidomes of three types of P. patens cells by mass spectrometry. In the present work, the antimicrobial activity of six of these peptides toward the gram-positive bacteria Bacillus subtilis SHgw and Clavibacter michiganensis pv. michiganensis and gram-negative bacteria Escherichia coli K12 and Xanthomonas arboricola 3004 has been revealed. The results have shown that three of six peptides inhibit the growth of the phytopathogenic bacteria X. arboricola and C. m. pv. michiganensis; four peptides inhibit the growth of the gram-negative bacterium E. coli K12, and one peptide inhibits the growth of the gram-positive bacterium B. subtilis. It has been found that the peptides inhibiting the bacterial growth are predominantly the fragments of ribosomal proteins. The work confirms the potential of the biological activity of peptides that are the degradation products of functional proteins.     

Melanization of <Emphasis Type="Italic">Fusarium keratoplasticum</Emphasis> (<Emphasis Type="Italic">F. solani</Emphasis> Species Complex) During Disseminated Fusariosis in a Patient with Acute Leukemia

Fusarium spp. are recognized as the second most frequently filamentous fungi causing opportunistic infections and particularly important due to the increasing number of immunocompromised patients. F. keratoplasticum (a member of F. solani species complex) is one of the Fusarium species commonly associated with human infection, and therefore, studies on the virulence of this fungus are needed. This study aimed to confirm the presence of melanin in F. keratoplasticum from a patient with systemic fusariosis. Immunofluorescence labeling with anti-melanin monoclonal antibody (MAb) was used to examine an expression of melanin in F. keratoplasticum in vitro and during infection. Electron spin resonance identified the particles extracted from F. keratoplasticum as stable free radical consistent with melanin. Lesional skin from the sites with fusariosis contained hyphal structures that could be labeled by melanin-binding MAb, while digestion of the tissue yielded dark particles that were reactive. These findings suggest that F. keratoplasticum hyphae and chlamydospores can produce melanin in vitro and that hyphae can synthesize pigment in vivo. Given the potential role of melanin in virulence of other fungi, this pigment in F. keratoplasticum may play a role in the pathogenesis of fusariosis.     

Expressing accessory proteins in cellulolytic <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> to improve the conversion yield of recalcitrant cellulose

Background

A recently constructed cellulolytic Yarrowia lipolytica is able to grow efficiently on an industrial organosolv cellulose pulp, but shows limited ability to degrade crystalline cellulose. In this work, we have further engineered this strain, adding accessory proteins xylanase II (XYNII), lytic polysaccharide monooxygenase (LPMO), and swollenin (SWO) from Trichoderma reesei in order to enhance the degradation of recalcitrant substrate.

Results

The production of EG I was enhanced using a promoter engineering strategy. This provided a new cellulolytic Y. lipolytica strain, which compared to the parent strain, exhibited higher hydrolytic activity on different cellulosic substrates. Furthermore, three accessory proteins, TrXYNII, TrLPMOA and TrSWO, were individually expressed in cellulolytic and non-cellulolytic Y. lipolytica. The amount of rhTrXYNII and rhTrLPMOA secreted by non-cellulolytic Y. lipolytica in YTD medium during batch cultivation in flasks was approximately 62 and 52 mg/L, respectively. The purified rhTrXYNII showed a specific activity of 532 U/mg-protein on beechwood xylan, while rhTrLPMOA exhibited a specific activity of 14.4 U/g-protein when using the Amplex Red/horseradish peroxidase assay. Characterization of rhTrLPMOA revealed that this protein displays broad specificity against β-(1,4)-linked glucans, but is inactive on xylan. Further studies showed that the presence of TrLPMOA synergistically enhanced enzymatic hydrolysis of cellulose by cellulases, while TrSWO1 boosted cellulose hydrolysis only when it was applied before the action of cellulases. The presence of rTrXYNII enhanced enzymatic hydrolysis of an industrial cellulose pulp and of wheat straw. Co-expressing TrXYNII and TrLPMOA in cellulolytic Y. lipolytica with enhanced EG I production procured a novel engineered Y. lipolytica strain that displayed enhanced ability to degrade both amorphous (CIMV-cellulose) and recalcitrant crystalline cellulose in complex biomass (wheat straw) by 16 and 90%, respectively.

Conclusions

This study has provided a potent cellulose-degrading Y. lipolytica strain that co-expresses a core set of cellulolytic enzymes and some accessory proteins. Results reveal that the tuning of cellulase production and the production of accessory proteins leads to optimized performance. Accordingly, the beneficial effect of accessory proteins for cellulase-mediated degradation of cellulose is underlined, especially when crystalline cellulose and complex biomass are used as substrates. Findings specifically underline the benefits and specific properties of swollenin. Although in our study swollenin clearly promoted cellulase action, its use requires process redesign to accommodate its specific mode of action.

     

Functional analysis of recombinant human and <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> <Emphasis Type="Italic">O</Emphasis>-GlcNAc transferases expressed in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation is an important post-translational modification in many cellular processes. It is mediated by O-GlcNAc transferases (OGTs), which catalyze the addition of O-GlcNAc to serine or threonine residues of the target proteins. In this study, we expressed a putative Yarrowia lipolytica OGT (YlOGT), the only homolog identified in the subphylum Saccharomycotina through bioinformatics analysis, and the human OGT (hOGT) as recombinant proteins in Saccharomyces cerevisiae, and performed their functional characterization. Immunoblotting assays using antibody against O-GlcNAc revealed that recombinant hOGT (rhOGT), but not the recombinant YlOGT (rYlOGT), undergoes auto-O-GlcNAcylation in the heterologous host S. cerevisiae. Moreover, the rhOGT expressed in S. cerevisiae showed a catalytic activity during in vitro assays using casein kinase II substrates, whereas no such activity was obtained in rYlOGT. However, the chimeric human-Y. lipolytica OGT, carrying the human tetratricopeptide repeat (TPR) domain along with the Y. lipolytica catalytic domain (CTD), mediated the transfer of O-GlcNAc moiety during the in vitro assays. Although the overexpression of full-length OGTs inhibited the growth of S. cerevisiae, no such inhibition was obtained upon overexpression of only the CTD fragment, indicating the role of TPR domain in growth inhibition. This is the first report on the functional analysis of the fungal OGT, indicating that the Y. lipolytica OGT retains its catalytic activity, although the physiological role and substrates of YlOGT remain to be elucidated.     

Early expression of WUSCHEL is a marker for in vitro shoot morphogenesis in tobacco and <Emphasis Type="Italic">Beta palonga</Emphasis>

We have studied the role of growth regulators behind in vitro shoot organogenesis and somatic embryogenesis in two plant systems, viz. tobacco (Nicotiana tabacum L. var. Jayasri) and Beta palonga R.K. Basu & K.K. Mukh. We have also correlated the phenomena of de differentiation with the relative expression of WUS (WUSCHEL) gene in a time-dependent manner. The results indicated that early WUS gene expression is a definite marker for in vitro shoot organogenesis in tobacco and Beta both in direct and indirect modes of regeneration. Additionally, we have performed a comparative homology modeling and in silico structural analysis of WUSCHEL proteins of B. palonga, B. vulgaris, and Arabidopsis to find out the commonality of the ligand binding site. The amino acids of the binding sites were identical (Arginine, Tryptophan, Proline, Asparagine, and Tyrosine) in the three materials under study; except two additional amino acids (Isoleucine and Alanine) in B. vulgaris.     

Anti-inflammatory activity of dimethyl octenol and oleanene tetrol isolated from <Emphasis Type="Italic">Trianthema decandra</Emphasis> L.

Dimethyl octenol from chloroform extract and oleanene tetrol from water extract of Trianthema decandra (TD) were isolated and characterized by using HPLC, UV, FT-IR, NMR, LC–MS and CHNS, their structure were elucidated from their respective spectral data. The anti-inflammatory activity of chloroform extract, water extract, dimethyl octenol and oleanene tetrol of T. decandra were studied and underlying cellular and molecular mechanisms of action were investigated in vitro and in vivo using macrophage-like cell line (RAW264.7 cells) and type II collagen induced arthritis mice models. Nitric oxide production was inhibited and TNF-α secretion was supressed in stimulated RAW cells treated with the chloroform extract and dimethyl octenol of T. decandra. Further, the chloroform and water extract, dimethyl octenol and oleanene tetrol inhibited protein denaturation and stabilized HRBC membranes in vitro. Reduction in inflammation as a measure of paw diameter was recorded in all the treated animals when compared to control animals. Catalase, peroxidase and glutathione peroxidase levels significantly increased in the joint tissue of treated groups. The possible mechanism of action of these compounds was studied using in silico molecular docking methods with phospholipase A₂ (PLA₂), cycloxygenase-1 (COX-1) and cycloxygenase-2 (COX-2) as targets. Among the three target proteins, the inhibition of the inflammatory protein PLA₂ and COX-2 towards dimethyl octenol and oleanene tetrol respectively. Our results contribute towards confirmation of the traditional use of TD and its compounds for the therapy of rheumatoid arthritis and other inflammatory joint disorders.     

Variable contributions of tyrosine residues to the structural and spectroscopic properties of the factor for inversion stimulation

The diverse roles of tyrosine residues in proteins may be attributed to their dual hydrophobic and polar nature, which can result in hydrophobic and ring stacking interactions, as well as hydrogen bonding. The small homodimeric DNA binding protein, factor for inversion stimulation (FIS), contains four tyrosine residues located at positions 38, 51, 69, and 95, each involved in specific intra- or intermolecular interactions. To investigate their contributions to the stability, flexibility, and spectroscopic properties of FIS, each one was independently mutated to phenylalanine. Equilibrium denaturation experiments show that Tyr95 and Tyr51 stabilize FIS by about 2 and 1 kcal/mol, respectively, as a result of their involvement in a hydrogen bond-salt bridge network. In contrast, Tyr38 destabilizes FIS by about 1 kcal/mol due to the placement of a hydroxyl group in a hydrophobic environment. The stability of FIS was not altered when the solvent-exposed Tyr69 was mutated. Limited proteolysis with trypsin and V8 proteases was used to monitor the flexibility of the C-terminus (residues 71-98) and the dimer core (residues 26-70), respectively. The results for Y95F and Y51F FIS revealed a different proteolytic susceptibility of the dimer core compared to the C-terminus, suggesting an increased flexibility of the latter. DNA binding affinity of the various FIS mutants was only modestly affected and correlated inversely with the C-terminal flexibility probed by trypsin proteolysis. Deconvolution of the fluorescence contribution of each mutant revealed that it varies in intensity and direction for each tyrosine in WT FIS, highlighting the role of specific interactions and the local environment in determining the fluorescence of tyrosine residues. The significant changes in stability, flexibility, and signals observed for the Y51F and Y95F mutations are attributed to their coupled participation in the hydrogen bond-salt bridge network. These results highlight the importance of tyrosine hydrogen-bonding and packing interactions for the stability of FIS and demonstrate the varying roles that tyrosine residues can play on the structural and spectroscopic properties of even small proteins.     

Characterization of the tyrosine recombinase MrpA encoded by the <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> A3(2) plasmid SCP2*

MrpA is the multimer resolution protein of the Streptomyces coelicolor A3(2) plasmid SCP2*. Previously, MrpA was found to significantly increase the stability of SCP2*-derived plasmids in Streptomyces lividans. The present report gives a functional characterization of MrpA. A sequence alignment revealed that MrpA shares highly conserved residues with members of the tyrosine recombinase family. After overexpression and Strep-tag purification, a DNase I footprint analysis and a gel mobility shift assay allowed for the identification of the 36-bp MrpA binding site mrpS. The mrpS site shows the configuration typical for tyrosine recombinases and contains two MrpA binding sites. The activity of MrpA was explored in vivo in E. coli cells and in vitro using purified MrpA. Depending on the position and orientation of the mrpS sites, three activities were detected: integration, resolution, and inversion. No accessory sites or proteins were required. Substitution of the conserved tyrosine (Y354F) by site-directed mutagenesis resulted in a complete loss of recombination activity but it still allowed the binding of MrpA to mrpS. The results define MrpA as a new site-specific tyrosine recombinase that acts with mrpS. In addition, we suggest that Y354 provides the nucleophile for DNA cleavage during recombination.