A versatile inhibitor of digestive enzymes in Aedes aegypti larvae selected from a pacifastin (TiPI) phage display library.

In Brazil, the major vector of arboviruses is Aedes aegypti, which can transmit several alpha and flaviviruses. In this work, a pacifastin protease inhibitor library was constructed and used to select mutants for Ae. aegypti larvae digestive enzymes. The library contained a total of 3.25 × 105 cfu with random mutations in the reactive site (P2-P2'). The most successfully selected mutant, TiPI6, a versatile inhibitor, was able to inhibit all three Ae. aegypti larvae proteolytic activities, trypsin-like, chymotrypsin-like and elastase-like activities, with IC50 values of 0.212 nM, 0.107 nM and 0.109 nM, respectively. In conclusion, the TiPI mutated phage display library was shown to be a useful tool for the selection of an inhibitor of proteolytic activities combined in a mix. TiPI6 is capable of controlling all three digestive enzyme activities present in the larval midgut extract. To our knowledge, this is the first time that one inhibitor containing a Gln at the P1 position showed inhibitory activity against trypsin, chymotrypsin, and elastase-like activities. TiPI6 can be a candidate for further larvicidal studies.

Structural analyses of a human lysyl-tRNA synthetase mutant associated with autosomal recessive nonsyndromic hearing impairment.

Aminoacyl-tRNA synthetases (AARSs) catalyze the ligation of amino acids to their cognate tRNAs and therefore play an essential role in protein biosynthesis in all living cells. The KARS gene in human encodes both cytosolic and mitochondrial lysyl-tRNA synthetase (LysRS). A recent study identified a missense mutation in KARS gene (c.517T > C) that caused autosomal recessive nonsyndromic hearing loss. This mutation led to a tyrosine to histidine (YH) substitution in both cytosolic and mitochondrial LysRS proteins, and decreased their aminoacylation activity to different levels. Here, we report the crystal structure of LysRS YH mutant at a resolution of 2.5 Å. We found that the mutation did not interfere with the active center, nor did it cause any significant conformational changes in the protein. The loops involved in tetramer interface and tRNA anticodon binding site showed relatively bigger variations between the mutant and wild type proteins. Considering the differences between the cytosolic and mitochondrial tRNAlyss, we suggest that the mutation triggered subtle changes in the tRNA anticodon binding region, and the interferences were further amplified by the different D and T loops in mitochondrial tRNAlys, and led to a complete loss of the aminoacylation of mitochondrial tRNAlys.

Dramatic Changes in Oligomerization Property Caused by Single Residue Deletion in Staphylococcus aureus Enolase.

Studies were conducted to understand the role of C-terminal lysine residues in the catalytic activity, structural stability and oligomeric properties of Staphylococcus aureus enolase. Interestingly, the S. aureus enolase, in solution, shows its presence as a stable dimer as well as the catalytically active fragile octamer. Compared to the hexa-histidine tagged S. aureus enolase (rSaeno), the deletion mutant showed the negligible difference in Km, but approximately 20-25% reduction in maximum reaction velocity (Vmax) and 2% reduction in turnover number were observed. These kinetic parameters indicate that K-434Δ deletion mutation does not drastically compromise the enzyme efficiency. The secondary structure and the octameric conformation of both the rSaeno and the K-434Δ mutant are very much stable between pH ranging from 6 to 9, temperatures ranging from 20 to 40 °C and in the presence of divalent metal ions Mg2+, Zn2+ and Mn2+. Under these conditions, the recombinant enzyme and the mutant are also catalytically very active. Intrinsic tryptophan fluorescence (320-380 nm) and CD spectral (195-260 nm) analysis revealed that the secondary structure and the surface architecture of the proteins are not majorly altered by the mutation. But, a significant correlation was observed between the time-dependent decrease in the catalytic activity and the oligomeric stability of rSaeno and K-434Δ mutant. The C-terminal lysine residues in the inter-dimer groove aid in folding and oligomerization of S. aureus enolase.

PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase.

Structural studies with tryptophan synthase (TS) bienzyme complex (α2ß2 TS) from Salmonella typhimurium have been performed to better understand its catalytic mechanism, allosteric behavior, and details of the enzymatic transformation of substrate to product in PLP-dependent enzymes. In this work, a novel expression system to produce the isolated α- and isolated ß-subunit allowed the purification of high amounts of pure subunits and α2ß2 StTS complex from the isolated subunits within 2 days. Purification was carried out by affinity chromatography followed by cleavage of the affinity tag, ammonium sulfate precipitation, and size exclusion chromatography (SEC). To better understand the role of key residues at the enzyme ß-site, site-direct mutagenesis was performed in prior structural studies. Another protocol was created to purify the wild type and mutant α2ß2 StTS complexes. A simple, fast and efficient protocol using ammonium sulfate fractionation and SEC allowed purification of α2ß2 StTS complex in a single day. Both purification protocols described in this work have considerable advantages when compared with previous protocols to purify the same complex using PEG 8000 and spermine to crystalize the α2ß2 StTS complex along the purification protocol. Crystallization of wild type and some mutant forms occurs under slightly different conditions, impairing the purification of some mutants using PEG 8000 and spermine. To prepare crystals suitable for x-ray crystallographic studies several efforts were made to optimize crystallization, crystal quality and cryoprotection. The methods presented here should be generally applicable for purification of tryptophan synthase subunits and wild type and mutant α2ß2 StTS complexes.

A High-Throughput Screening System Based on Droplet Microfluidics for Glucose Oxidase Gene Libraries.

Glucose oxidase (GOx) is an important industrial enzyme that can be optimized for specific applications by mutagenesis and activity-based screening. To increase the efficiency of this approach, we have developed a new ultrahigh-throughput screening platform based on a microfluidic lab-on-chip device that allows the sorting of GOx mutants from a saturation mutagenesis library expressed on the surface of yeast cells. GOx activity was measured by monitoring the fluorescence of water microdroplets dispersed in perfluorinated oil. The signal was generated via a series of coupled enzyme reactions leading to the formation of fluorescein. Using this new method, we were able to enrich the yeast cell population by more than 35-fold for GOx mutants with higher than wild-type activity after two rounds of sorting, almost double the efficiency of our previously described flow cytometry platform. We identified and characterized novel GOx mutants, the most promising of which (M6) contained a combination of six point mutations that increased the catalytic constant kcat by 2.1-fold compared to wild-type GOx and by 1.4-fold compared to a parental GOx variant. The new microfluidic platform for GOx was therefore more sensitive than flow cytometry and supports comprehensive screens of gene libraries containing multiple mutations per gene.

The carboxy-terminal insert in the Q-loop is needed for functionality of Escherichia coli cytochrome bd-I.

Cytochrome bd, a component of the prokaryotic respiratory chain, is important under physiological stress and during pathogenicity. Electrons from quinol substrates are passed on via heme groups in the CydA subunit and used to reduce molecular oxygen. Close to the quinol binding site, CydA displays a periplasmic hydrophilic loop called Q-loop that is essential for quinol oxidation. In the carboxy-terminal part of this loop, CydA from Escherichia coli and other proteobacteria harbors an insert of ~60 residues with unknown function. In the current work, we demonstrate that growth of the multiple-deletion strain E. coli MB43∆cydA (∆cydA∆cydB∆appB∆cyoB∆nuoB) can be enhanced by transformation with E. coli cytochrome bd-I and we utilize this system for assessment of Q-loop mutants. Deletion of the cytochrome bd-I Q-loop insert abolished MB43∆cydA growth recovery. Swapping the cytochrome bd-I Q-loop for the Q-loop from Geobacillus thermodenitrificans or Mycobacterium tuberculosis CydA, which lack the insert, did not enhance the growth of MB43∆cydA, whereas swapping for the Q-loop from E. coli cytochrome bd-II recovered growth. Alanine scanning experiments identified the cytochrome bd-I Q-loop insert regions Ile318-Met322, Gln338-Asp342, Tyr353-Leu357, and Thr368-Ile372 as important for enzyme functionality. Those mutants that completely failed to recover growth of MB43∆cydA also lacked oxygen consumption activity and heme absorption peaks. Moreover, we were not able to isolate cytochrome bd-I from these inactive mutants. The results indicate that the cytochrome bd Q-loop exhibits low plasticity and that the Q-loop insert in E. coli is needed for complete, stable, assembly of cytochrome bd-I.

Improved Electrophoretic Separation to Assist the Monitoring of Bcl-xL Post-Translational Modifications.

Bcl-xL is an oncogene of which the survival functions are finely tuned by post-translational modifications (PTM). Within the Bcl-2 family of proteins, Bcl-xL shows unique eligibility to deamidation, a time-related spontaneous reaction. Deamidation is still a largely overlooked PTM due to a lack of easy techniques to monitor Asn→Asp/IsoAsp conversions or Glu→Gln conversions. Being able to detect PTMs is essential to achieve a comprehensive description of all the regulatory mechanisms and functions a protein can carry out. Here, we report a gel composition improving the electrophoretic separation of deamidated forms of Bcl-xL generated either by mutagenesis or by alkaline treatment. Importantly, this new gel formulation proved efficient to provide the long-sought evidence that even doubly-deamidated Bcl-xL remains eligible for regulation by phosphorylation.

Temperature-Independent Kinetic Isotope Effects as Evidence for a Marcus-like Model of Hydride Tunneling in Phosphite Dehydrogenase.

Phosphite dehydrogenase catalyzes the transfer of a hydride from phosphite to NAD+, producing phosphate and NADH. We have evaluated the role of hydride tunneling in a thermostable variant of this enzyme (17X-PTDH) by measuring the temperature dependence of the primary 2H kinetic isotope effects (KIEs) between 5 and 45 °C. Pre-steady-state kinetic measurements were used to demonstrate that the hydride transfer is rate-determining across this temperature range and that the observed KIEs are equal to the intrinsic isotope effect on the chemical step. The KIEs on the pre-exponential factor (AH/AD) and the activation energy (ΔEa) were 1.6 ± 0.1 and 0.21 ± 0.05 kcal/mol, respectively, suggesting that 17X-PTDH facilitates extensive tunneling of both isotopes via a Marcus-like model. Site-directed mutagenesis was used to evaluate the role of an active site threonine (Thr104) found on the back face of the nicotinamide in promoting the close packing of the substrates. In mutants with reduced steric bulk at this position, values of AH/AD and ΔEa fall within the range describing semiclassical "over the barrier" reactivity, suggesting that Thr104 acts as a steric backstop to promote tunneling in 17X-PTDH. Whereas hydrogen tunneling is now a widely appreciated feature of C-H activating enzymes, these observations with a P-H activating system are consistent with the proposal that tunneling is likely to be a common feature on all enzymes that catalyze hydrogen transfers.

Characterization of a novel multi-domain xylanase from Clostridium clariflavum with application in hydrolysis of corn cobs.

OBJECTIVES: To develop a novel multi-catalytic domain (CD) xylanase Xyn2083 from Clostridium clariflavum by expression of its truncated forms in Escherichia coli and cooperation of xylanase with cellulase in the hydrolysis of waste lignocellulosic resources. RESULTS: Xyn2083 has two glycoside hydrolase family (GH) domains GH11 and GH10. These two catalytic domains functioned synergistically in xylan hydrolysis. The recombinant protein with GH11 domain, Xyn2083GH11, had the highest xylanase activity among three constructed truncated forms. The deletion of N-terminal extra amino acid residues of Xyn2083GH11 decreased catalytic activity as well as the stability of the enzyme. The hydrolysis rates of cellulose and xylan in the pretreated corn cobs were 90.56% and 72.80% with the addition of Xyn2083GH11 and cellulase, whereas those were 67.95% and 34.45% using sole cellulase respectively. The structural analysis of substrates indicated that the addition of Xyn2083GH11 led to a looser structure and more exposure of crystal cellulose for cellulase to approach. CONCLUSIONS: Since the native multi-CDs' xylanases are rare, the thermostable Xyn2083 provides a good source for functional studies of two CDs coexisted in one xylanase and for potential applications after modification.

The molecular mechanism of dsRNA processing by a bacterial Dicer.

Members of the ribonuclease (RNase) III family regulate gene expression by processing dsRNAs. It was previously shown that Escherichia coli (Ec) RNase III recognizes dsRNA with little sequence specificity and the cleavage products are mainly 11 nucleotides (nt) long. It was also shown that the mutation of a glutamate (EcE38) to an alanine promotes generation of siRNA-like products typically 22 nt long. To fully characterize substrate specificity and product size of RNase IIIs, we performed in vitro cleavage of dsRNAs by Ec and Aquifex aeolicus (Aa) enzymes and delineated their products by next-generation sequencing. Surprisingly, we found that both enzymes cleave dsRNA at preferred sites, among which a guanine nucleotide was enriched at a specific position (+3G). Based on sequence and structure analyses, we conclude that RNase IIIs recognize +3G via a conserved glutamine (EcQ165/AaQ161) side chain. Abolishing this interaction by mutating the glutamine to an alanine eliminates the observed +3G preference. Furthermore, we identified a second glutamate (EcE65/AaE64), which, when mutated to alanine, also enhances the production of siRNA-like products. Based on these findings, we created a bacterial Dicer that is ideally suited for producing heterogeneous siRNA cocktails to be used in gene silencing studies.

Establishment of a high throughput-screening system for nucleoside deoxyribosyltransferase II mutant enzymes with altered substrate specificity.

Nucleoside deoxyribosyltransferase II (NDT) catalyzes the transglycosylation reaction of the 2'-deoxyribose moiety between purine and/or pyrimidine bases and has been widely used in the synthesis of nucleoside analogs. The high specificity of NDT for 2'-deoxyribose limits its applications. Because 2'C- and/or 3'C-modified nucleosides have been widely used as antiviral or antitumour agents, improving the activity of NDT towards these modified nucleosides by protein engineering is an area of interest to the pharmaceutical industry. NDT engineering is hindered by a lack of effective screening methods. This study developed a high-throughput screening system, which was established by nucleoside deoxyribosyltransferase II-cytidine deaminase co-expression, indophenol colorimetric assay and whole-cell catalysis. A high-throughput screening system for NDT was established for the first time. This system can be applied to detect NDT-specific activity for a variety of cytidine analogs with glycosyl and base modifications, such as 5-aza-2'-deoxycytidine, 2',3'-dideoxycytidine, cytosine-ß-d-arabinofuranoside. In this study, we adopted the semi-rational design of NDT and constructed a mutant library of NDT from Lactobacillus helveticus (LhNDT) by site-saturation mutagenesis. Over 600 mutants were screened, and a variant with up to a 5.2-fold higher conversion rate of 2',3'-dideoxyinosine was obtained.

Identification of Thermus aquaticus DNA polymerase variants with increased mismatch discrimination and reverse transcriptase activity from a smart enzyme mutant library.

DNA polymerases the key enzymes for several biotechnological applications. Obviously, nature has not evolved these enzymes to be compatible with applications in biotechnology. Thus, engineering of a natural scaffold of DNA polymerases may lead to enzymes improved for several applications. Here, we investigated a two-step approach for the design and construction of a combinatorial library of mutants of KlenTaq DNA polymerase. First, we selected amino acid sites for saturation mutagenesis that interact with the primer/template strands or are evolutionarily conserved. From this library, we identified mutations that little interfere with DNA polymerase activity. Next, these functionally active mutants were combined randomly to construct a second library with enriched sequence diversity. We reasoned that the combination of mutants that have minuscule effect on enzyme activity and thermostability, will result in entities that have an increased mutation load but still retain activity. Besides activity and thermostability, we screened the library for entities with two distinct properties. Indeed, we identified two different KlenTaq DNA polymerase variants that either exhibit increased mismatch extension discrimination or increased reverse transcription PCR activity, respectively.

Proteome-wide onco-proteogenomic somatic variant identification in ER-positive breast cancer.

BACKGROUND: Recent advances in mass spectrometric instrumentation and bioinformatics have critically contributed to the field of proteogenomics. Nonetheless, whether that integrative approach has reached the point of maturity to effectively reveal the flow of genetic variants from DNA to proteins still remains elusive. The objective of this study was to detect somatically acquired protein variants in breast cancer specimens for which full genome and transcriptome data was already available (BASIS cohort). METHODS: LC-MS/MS shotgun proteomic results of 21 breast cancer tissues were coupled to DNA sequencing data to identify variants at the protein level and finally were used to associate protein expression with gene expression levels. RESULTS: Here we report the observation of three sequencing-predicted single amino acid somatic variants. The sensitivity of single amino acid variant (SAAV) detection based on DNA sequencing-predicted single nucleotide variants was 0.4%. This sensitivity was increased to 0.6% when all the predicted variants were filtered for MS "compatibility" and was further increased to 2.9% when only proteins with at least one wild type peptide detected were taken into account. A correlation of mRNA abundance and variant peptide detection revealed that transcripts for which variant proteins were detected ranked among the top 6.3% most abundant transcripts. The variants were detected in highly abundant proteins as well, thus establishing transcript and protein abundance and MS "compatibility" as the main factors affecting variant onco-proteogenomic identification. CONCLUSIONS: While proteomics fails to identify the vast majority of exome DNA variants in the resulting proteome, its ability to detect a small subset of SAAVs could prove valuable for precision medicine applications.

Design and assessment of an active anti-epidermal growth factor receptor (EGFR) single chain variable fragment (ScFv) with improved solubility.

ScFv is emerging as a therapeutic alternative to the full-length monoclonal antibodies due to its small size and low production cost, but its low solubility remains a limiting factor toward wider use. Here, we increased the solubility of an Anti-epidermal growth factor receptor ScFv (Anti-EGFR ScFv) by attaching, a short 12-residue solubility enhancing peptide (SEP) tag at its C terminus. We first estimated the solubility increase by running 500-ns Brownian dynamics (BD) simulations. We then experimentally evaluated the predictions by producing recombinant Anti-EGFR ScFv with and without a SEP tag (called C9R) in E. coli. At 20 °C, ∼85% of Anti-EGFR ScFv-C9R expressed in the soluble fraction, whereas all of the Anti-EGFR ScFv remained in the insoluble fraction. The total yield of Anti-EGFR ScFv-C9R was 17.15 mg which was ∼3 times higher than that of Anti-EGFR ScFv refolded from the insoluble fraction. Static and dynamic light scattering demonstrated the higher solubility of the purified Anti-EGFR ScFv-C9R, and Circular Dichroism (CD) indicated its high thermal stability, whereas the untagged protein aggregated at 37 °C and pH 6. Finally, the binding activity of Anti-EGFR ScFv-C9R to EGFR was confirmed by surface plasmon resonance (SPR). Altogether, these results illustrate the improved biophysical and biochemical characteristics of Anti-EGFR ScFv-C9R and emphasize the potentials of SEP-tags for enhancing the solubility of aggregation-prone antibody fragments.

Enhanced trypsin thermostability in Pichia pastoris through truncating the flexible region.

BACKGROUND: High thermostability is required for trypsin to have wider industrial applications. Target mutagenesis at flexible regions has been proved to be an efficient protein engineering method to enhance the protein thermostability. RESULTS: The flexible regions in porcine trypsin were predicted using the methods including molecular dynamic simulation, FlexPred, and FoldUnfold. The amino acids 78-90 was predicted to be the highly flexible region simultaneously by the three methods and hence selected to be the mutation target. We constructed five variants (D3, D5, D7, D9, and D11) by truncating the region. And the variant D9 showed higher thermostability, with a 5 °C increase in Topt, 5.8 °C rise in [Formula: see text], and a 4.5 °C rise in Tm, compared to the wild-type. Moreover, the half-life value of the variant D9 was also found to be dramatically improved by 46 min. Circular dichroism and intrinsic fluorescence indicated that the structures had no significant change between the variant D9 and the wild-type. The surface hydrophobicity of D9 was measured to be lower than that of wild-type, indicating the increased hydrophobic interaction, which could have contributed to the improved thermostability of D9. CONCLUSIONS: These results showed that the thermostability of variant D9 was increased. The variant D9 could be expected to be a promising tool enzyme for its wider industrial applications. The method of truncating the flexible region used in our study has the potential to be used for enhancing the thermostability of other proteins.

Protein stabilization with retained function of monellin using a split GFP system.

Sweet proteins are an unexploited resource in the search for non-carbohydrate sweeteners mainly due to their low stability towards heating. Variants of the sweet protein monellin, with increased stability, were derived by an in vivo screening method based on the thermodynamic linkage between fragment complementation and protein stability. This approach depends on the correlation between mutational effects on the affinity between protein fragments and the stability of the intact protein. By linking the two fragments of monellin to the split GFP (green fluorescent protein) system, reconstitution of GFP was promoted and moderately fluorescent colonies were obtained. Two separate random libraries were produced for the monellin chains and the mutant clones were ranked based on fluorescence intensity. Mutants with increased affinity between the fragments, and subsequently increased stability, caused increased fluorescence intensity of split GFP. Single chain monellin variants of the top-ranked mutants for each chain, S76Y in the A-chain and W3C + R39G in the B-chain and all combinations thereof, were expressed and the increase in stability was verified by temperature denaturation studies using circular dichroism spectroscopy. Functionality studies showed that mutant S76Y has retained sweetness and has potential use within the food industry.

A novel strategy to improve the thermostability of Penicillium camembertii mono- and di-acylglycerol lipase.

Penicillium camembertii (PCL), a mono- and di-acylglycerol lipase (DGL), has the vital potential in the oil chemistry for food industry. However, known DGLs are mesophilic enzymes which restricts its application in the industry. To improve thermostability of PCL, we used amino acid substitution by comparison of amino acids compositions of PCL and protein sequences from typical thermophilic bacteria. Then, some conservative residues around active center were avoided to mutate according to homologous alignment analyses. Furthermore, the list was narrowed down to 28 candidate mutational sites of PCL by analyzing the hydrophobic interaction of amino acids in the structure. And among them only the mutant PCL-D25R had formed an additional salt bridge between R25-D32 and increased more hydrogen bonds interaction. Therefore, mutant PCL-D25R were constructed and expressed. Thermal inactivation assay showed that the half-life of mutant PCL-D25R at 45 °C increased 4-fold compared to that of PCL-WT. Melting temperature of mutant PCL-D25R increased to 49.5 °C from 46.5 °C by fluorescence-based thermal stability assay. This study provides a valuable strategy for engineering DGL thermostability.

A chemometric approach for characterization of serum transthyretin in familial amyloidotic polyneuropathy type I (FAP-I) by electrospray ionization-ion mobility mass spectrometry.

In this study, we describe a chemometric data analysis approach to assist in the interpretation of the complex datasets from the analysis of high-molecular mass oligomeric proteins by ion mobility mass spectrometry (IM-MS). The homotetrameric protein transthyretin (TTR) is involved in familial amyloidotic polyneuropathy type I (FAP-I). FAP-I is associated with a specific TTR mutant variant (TTR(Met30)) that can be easily detected analyzing the monomeric forms of the mutant protein. However, the mechanism of protein misfolding and aggregation onset, which could be triggered by structural changes in the native tetrameric protein, remains under investigation. Serum TTR from healthy controls and FAP-I patients was purified under non-denaturing conditions by conventional immunoprecipitation in solution and analyzed by IM-MS. IM-MS allowed separation and characterization of several tetrameric, trimeric and dimeric TTR gas ions due to their differential drift time. After an appropriate data pre-processing, multivariate curve resolution alternating least squares (MCR-ALS) was applied to the complex datasets. A group of seven independent components being characterized by their ion mobility profiles and mass spectra were resolved to explain the observed data variance in control and patient samples. Then, principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were considered for exploration and classification. Only four out of the seven resolved components were enough for an accurate differentiation. Furthermore, the specific TTR ions identified in the mass spectra of these components and the resolved ion mobility profiles provided a straightforward insight into the most relevant oligomeric TTR proteoforms for the disease.

Role of SH3b binding domain in a natural deletion mutant of Kayvirus endolysin LysF1 with a broad range of lytic activity.

The spontaneous host-range mutants 812F1 and K1/420 are derived from polyvalent phage 812 that is almost identical to phage K, belonging to family Myoviridae and genus Kayvirus. Phage K1/420 is used for the phage therapy of staphylococcal infections. Endolysin of these mutants designated LysF1, consisting of an N-terminal cysteine-histidine-dependent aminohydrolase/peptidase (CHAP) domain and C-terminal SH3b cell wall-binding domain, has deleted middle amidase domain compared to wild-type endolysin. In this work, LysF1 and both its domains were prepared as recombinant proteins and their function was analyzed. LysF1 had an antimicrobial effect on 31 Staphylococcus species of the 43 tested. SH3b domain influenced antimicrobial activity of LysF1, since the lytic activity of the truncated variant containing the CHAP domain alone was decreased. The results of a co-sedimentation assay of SH3b domain showed that it was able to bind to three types of purified staphylococcal peptidoglycan 11.2, 11.3, and 11.8 that differ in their peptide bridge, but also to the peptidoglycan type 11.5 of Streptococcus uberis, and this capability was verified in vivo using the fusion protein with GFP and fluorescence microscopy. Using several different approaches, including NMR, we have not confirmed the previously proposed interaction of the SH3b domain with the pentaglycine bridge in the bacterial cell wall. The new naturally raised deletion mutant endolysin LysF1 is smaller than LysK, has a broad lytic spectrum, and therefore is an appropriate enzyme for practical use. The binding spectrum of SH3b domain covering all known staphylococcal peptidoglycan types is a promising feature for creating new chimeolysins by combining it with more effective catalytic domains.

Design and characterization of new ß-glucuronidase active site variants with altered substrate specificity.

OBJECTIVE: To isolate and characterize the kinetics of variants of E. coli ß-glucuronidase (GUS) having altered substrate specificity. RESULTS: Two small combinatorial libraries of E. coli GUS variants were constructed and screened for improved activities towards the substrate p-nitrophenyl-ß-D-galactoside (pNP-gal). Nine of the most active variants were purified and their kinetic parameters were determined. These variants show up to 134-fold improved kcat/KM value towards pNP-gal compared to wild-type GUS, up to 9 × 108-fold shift in specificity from p-nitrophenyl-ß-D-glucuronide (pNP-glu) to pNP-gal compared to wild-type, and 103-fold increase in specificity shift compared to a previously evolved GUS variant. CONCLUSIONS: The kinetic data collected for nine new GUS variants is invaluable for training computational protein design models that better predict amino acid substitutions which improve activity of enzyme variants having altered substrate specificity.