Engineering Streptomyces clavuligerus Deacetoxycephalosporin C Synthase for Optimal Ring Expansion Activity toward Penicillin G

The deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was engineered with the aim of enhancing the conversion of penicillin G into phenylacetyl-7-aminodeacetoxycephalosporanic acid, a precursor of 7-aminodeacetoxycephalosporanic acid, for industrial application. A single round of random mutagenesis followed by the screening of 5,500 clones identified three mutants, G79E, V275I, and C281Y, that showed a two- to sixfold increase in the k_cat/K_m ratio compared to the wild-type enzyme. Site-directed mutagenesis to modify residues surrounding the substrate resulted in three mutants, N304K, I305L, and I305M, with 6- to 14-fold-increased k_cat/K_m values. When mutants containing all possible combinations of these six sites were generated to optimize the ring expansion activity for penicillin G, the double mutant, YS67 (V275I, I305M), showed a significant 32-fold increase in the k_cat/K_m ratio and a 5-fold increase in relative activity for penicillin G, while the triple mutant, YS81 (V275I, C281Y, I305M), showed an even greater 13-fold increase in relative activity toward penicillin G. Our results demonstrate that this is a robust approach to the modification of DAOCS for an optimized DAOCS-penicillin G reaction.     

Directed Evolution of Streptomyces clavuligerus Deacetoxycephalosporin C Synthase for Enhancement of Penicillin G Expansion

The deacetoxycephalosporin C synthase from Streptomyces clavuligerus was directly modified for enhancement of penicillin G expansion into phenylacetyl-7-aminodeacetoxycephalosporanic acid, an important intermediate in the industrial manufacture of cephalosporin antibiotics. Nine new mutants, mutants M73T, T91A, A106T, C155Y, Y184H, M188V, M188I, H244Q, and L277Q with 1.4- to 5.7-fold increases in the k_cat/K_m ratio, were obtained by screening 6,364 clones after error-prone PCR-based random mutagenesis. Subsequently, DNA shuffling was carried out to screen possible combinations of substitutions, including previous point mutations. One quaternary mutant, the C155Y/Y184H/V275I/C281Y mutant, which had a k_cat/K_m ratio that was 41-fold higher was found after 10,572 clones were assayed. The distinct mutants obtained using different mutagenesis methods demonstrated the complementarity of the techniques. Interestingly, most of the mutated residues that result in enhanced activities are located within or near the unique small barrel subdomain, suggesting that manipulation of this subdomain may be a constructive strategy for improvement of penicillin expansion. Several mutations had very distinct effects on expansion of penicillins N and G, perhaps due to different penicillin-interacting modes within the enzyme. Thus, the present study provided not only promising enzymes for cephalosporin biosynthesis but also a large number of mutants, which provided new insights into the structure-function relationship of the protein that should lead to further rational engineering.     

A Complete Library of Amino Acid Alterations at N304 in Streptomyces clavuligerus Deacetoxycephalosporin C Synthase Elucidates the Basis for Enhanced Penicillin Analogue Conversion

N304 of Streptomyces clavuligerus deacetoxycephalosporin C synthase was mutagenized to alter its catalytic ability. Given that N304A, N304K, N304L, and N304R mutant enzymes exhibited significant improvements in penicillin analogue conversions, we advocate that replacement of N304 with residues with aliphatic or basic side chains is preferable for engineering of a hypercatalytic enzyme.     

Improved Infrared-Sensing Running Wheel Systems with an Effective Exercise Activity Indicator

This paper describes an infrared-sensing running wheel (ISRW) system for the quantitative measurement of effective exercise activity in rats. The ISRW system provides superior exercise training compared with commercially available traditional animal running platforms. Four infrared (IR) light-emitting diode/detector pairs embedded around the rim of the wheel detect the rat’s real-time position; the acrylic wheel has a diameter of 55 cm and a thickness of 15 cm, that is, it is larger and thicker than traditional exercise wheels, and it is equipped with a rubber track. The acrylic wheel hangs virtually frictionless, and a DC motor with an axially mounted rubber wheel, which has a diameter of 10 cm, drives the acrylic wheel from the outer edge. The system can automatically train rats to run persistently. The proposed system can determine effective exercise activity (EEA), with the IR sensors (which are connected to a conventional PC) recording the rat exercise behavior. A prototype of the system was verified by a hospital research group performing ischemic stroke experiments on rats by considering middle cerebral artery occlusion. The experimental data demonstrated that the proposed system provides greater neuroprotection in an animal stroke model compared with a conventional treadmill and a motorized running wheel for a given exercise intensity. The quantitative exercise effectiveness indicator showed a 92% correlation between an increase in the EEA and a decrease in the infarct volume. This indicator can be used as a noninvasive and objective reference in clinical animal exercise experiments.     

AdaptivSearch: A Novel Iterative Algorithm for the Generation of n-dimensional Hypersurfaces Using an Adaptive Numerical Strategy

AdaptivSearch is the first adaptive strategy based algorithm for the rational and economical construction of n-dimensional hypersurfaces. AdaptivSearch works iteratively: At each step it parcels out the definition range into several triangles, evaluates the worst according to a built-in error criterion, and refines the approximation to the unknown function by choosing the barycenter of this partial area as the node to be calculated next. Based upon the error criterion, AdaptivSearch selectively approaches those parts of the hypersurface in which the curvature exhibits the strongest changes. Some examples of AdaptivSearch applications for both analytical functions and chemical model surfaces are given in order to demonstrate the behavior of the algorithm. These show its broad applicability and the usefulness, especially for chemical problems.     

Disabling Mitochondrial Peroxide Metabolism via Combinatorial Targeting of Peroxiredoxin 3 as an Effective Therapeutic Approach for Malignant Mesothelioma

Dysregulation of signaling pathways and energy metabolism in cancer cells enhances production of mitochondrial hydrogen peroxide that supports tumorigenesis through multiple mechanisms. To counteract the adverse effects of mitochondrial peroxide many solid tumor types up-regulate the mitochondrial thioredoxin reductase 2 - thioredoxin 2 (TRX2) - peroxiredoxin 3 (PRX3) antioxidant network. Using malignant mesothelioma cells as a model, we show that thiostrepton (TS) irreversibly disables PRX3 via covalent crosslinking of peroxidatic and resolving cysteine residues in homodimers, and that targeting the oxidoreductase TRX2 with the triphenylmethane gentian violet (GV) potentiates adduction by increasing levels of disulfide-bonded PRX3 dimers. Due to the fact that activity of the PRX3 catalytic cycle dictates the rate of adduction by TS, immortalized and primary human mesothelial cells are significantly less sensitive to both compounds. Moreover, stable knockdown of PRX3 reduces mesothelioma cell proliferation and sensitivity to TS. Expression of catalase in shPRX3 mesothelioma cells restores defects in cell proliferation but not sensitivity to TS. In a SCID mouse xenograft model of human mesothelioma, administration of TS and GV together reduced tumor burden more effectively than either agent alone. Because increased production of mitochondrial hydrogen peroxide is a common phenotype of malignant cells, and TS and GV are well tolerated in mammals, we propose that targeting PRX3 is a feasible redox-dependent strategy for managing mesothelioma and other intractable human malignancies.     

Formate as an Auxiliary Substrate for Glucose-Limited Cultivation of Penicillium chrysogenum: Impact on Penicillin G Production and Biomass Yield

Production of β-lactams by the filamentous fungus Penicillium chrysogenum requires a substantial input of ATP. During glucose-limited growth, this ATP is derived from glucose dissimilation, which reduces the product yield on glucose. The present study has investigated whether penicillin G yields on glucose can be enhanced by cofeeding of an auxiliary substrate that acts as an energy source but not as a carbon substrate. As a model system, a high-producing industrial strain of P. chrysogenum was grown in chemostat cultures on mixed substrates containing different molar ratios of formate and glucose. Up to a formate-to-glucose ratio of 4.5 mol·mol⁻¹, an increasing rate of formate oxidation via a cytosolic NAD⁺-dependent formate dehydrogenase increasingly replaced the dissimilatory flow of glucose. This resulted in increased biomass yields on glucose. Since at these formate-to-glucose ratios the specific penicillin G production rate remained constant, the volumetric productivity increased. Metabolic modeling studies indicated that formate transport in P. chrysogenum does not require an input of free energy. At formate-to-glucose ratios above 4.5 mol·mol⁻¹, the residual formate concentrations in the cultures increased, probably due to kinetic constraints in the formate-oxidizing system. The accumulation of formate coincided with a loss of the coupling between formate oxidation and the production of biomass and penicillin G. These results demonstrate that, in principle, mixed-substrate feeding can be used to increase the yield on a carbon source of assimilatory products such as β-lactams.     

Site Directed Mutagenesis of Schizosaccharomyces pombe Glutathione Synthetase Produces an Enzyme with Homoglutathione Synthetase Activity

Three different His-tagged, mutant forms of the fission yeast glutathione synthetase (GSH2) were derived by site-directed mutagenesis. The mutant and wild-type enzymes were expressed in E. coli DH5α and affinity purified in a two-step procedure. Analysis of enzyme activity showed that it was possible to shift the substrate specificity of GSH2 from Gly (k_m 0,19; wild-type) to β-Ala or Ser. One mutation (substitution of Ile471, Cy472 to Met and Val and Ala 485 and Thr486 to Leu and Pro) increased the affinity of GSH2 for β-Ala (k_m 0,07) and lowered the affinity for Gly (k_m 0,83), which is a characteristic of the enzyme homoglutathione synthetase found in plants. Substitution of Ala485 and Thr486 to Leu and Pro only, increased instead the affinity of GSH2 for Ser (k_m 0,23) as a substrate, while affinity to Gly was preserved (k_m 0,12). This provides a new biosynthetic pathway for hydroxymethyl glutathione, which is known to be synthesized from glutathione and Ser in a reaction catalysed by carboxypeptidase Y. The reported findings provide further insight into how specific amino acids positioned in the GSH2 active site facilitate the recognition of different amino acid substrates, furthermore they support the evolutionary theory that homoglutathione synthetase evolved from glutathione synthetase by a single gene duplication event.     

Generation of an antibody with a designed specificity difference for protein C and activated protein C

Rabbit polyclonal antibodies to a synthetic peptide, NH₂-Asp-Thr-Asn-Gln-Val-Asp-Gln-Lys-Asp-Gln-Leu-Asp-Phe-Arg-CONH₂ (APep), have been produced. This sequence is identical to that contained in the tetradecapeptide released from bovine protein C (PC) as a result of its conversion to its activated form (APC), except that Phe₁₃ replaced the normal Pro₁₃, in order to discourage cross-reactivity of antibodies to the carboxylterminal portion of APep with PC. The antibody pool obtained reacted with PC and showed virtually no cross-reactivity toward either APC or several typical plasma proteins. This general approach should serve well as a means of production of antibodies with a designed specificity capable of distinguishing between forms of the same protein that arise by release of peptide material.     

Mutagenesis and phenotypic selection as a strategy toward domestication of Chlamydomonas reinhardtii strains for improved performance in photobioreactors

Microalgae have a valuable potential for biofuels production. As a matter of fact, algae can produce different molecules with high energy content, including molecular hydrogen (H(2)) by the activity of a chloroplastic hydrogenase fueled by reducing power derived from water and light energy. The efficiency of this reaction, however, is limited and depends from an intricate relationships between oxygenic photosynthesis and mitochondrial respiration. The way toward obtaining algal strains with high productivity in photobioreactors requires engineering of their metabolism at multiple levels in a process comparable to domestication of crops that were derived from their wild ancestors through accumulation of genetic traits providing improved productivity under conditions of intensive cultivation as well as improved nutritional/industrial properties. This holds true for the production of any biofuels from algae: there is the need to isolate multiple traits to be combined and produce organisms with increased performances. Among the different limitations in H(2) productivity, we identified three with a major relevance, namely: (i) the light distribution through the mass culture; (ii) the strong sensitivity of the hydrogenase to even very low oxygen concentrations; and (iii) the presence of alternative pathways, such as the cyclic electron transport, competing for reducing equivalents with hydrogenase and H(2) production. In order to identify potentially favorable mutations, we generated a collection of random mutants in Chlamydomonas reinhardtii which were selected through phenotype analysis for: (i) a reduced photosynthetic antenna size, and thus a lower culture optical density; (ii) an altered photosystem II activity as a tool to manipulate the oxygen concentration within the culture; and (iii) State 1-State 2 transition mutants, for a reduced cyclic electron flow and maximized electrons flow toward the hydrogenase. Such a broad approach has been possible thanks to the high throughput application of absorption/fluorescence optical spectroscopy methods. Strong and weak points of this approach are discussed.     

Purine Based Combinatorial Chemistry: Solution Phase Simultaneous Addition of Functionalities. Iterative Deconvolution by Orthogonal Protection to a Single Compound with Potent Antibacterial Activity

Abstract

We have recently described a method to prepare combinatorial chemistry libraries by solution phase simultaneous addition of functionalities (SPSAF).^1–2 SPSAF has been used to create libraries based on the purine heterocycle. The nucleophilic sites (secondary nitrogens) in the planer heteroaromatic purine scaffold were built in via linkers. Thus, to continue the use of electophilic functionalities, as in previous libraries, a bifunctional nucleophilic linker was required. Piperazines readily served this purpose. Nucleophilic displacement of the chloro groups on 2,6-dichloropurine with piperazines provides reactive, constrained secondary amines for combinatorialization (Figure 1, 1). An additional piperazine was placed in the 9-position by alkylation of 2,6-dipiperazinylpurine. In this manner, the functionality that differentiates each pool (sublibrary) could be placed last in the synthetic scheme (fix last concept).¹     

Use of Limited Proteolysis and Mutagenesis To Identify Folding Domains and Sequence Motifs Critical for Wax Ester Synthase/Acyl Coenzyme A:Diacylglycerol Acyltransferase Activity

Triacylglycerols and wax esters are synthesized as energy storage molecules by some proteobacteria and actinobacteria under stress. The enzyme responsible for neutral lipid accumulation is the bifunctional wax ester synthase/acyl-coenzyme A (CoA):diacylglycerol acyltransferase (WS/DGAT). Structural modeling of WS/DGAT suggests that it can adopt an acyl-CoA-dependent acyltransferase fold with the N-terminal and C-terminal domains connected by a helical linker, an architecture demonstrated experimentally by limited proteolysis. Moreover, we found that both domains form an active complex when coexpressed as independent polypeptides. The structural prediction and sequence alignment of different WS/DGAT proteins indicated catalytically important motifs in the enzyme. Their role was probed by measuring the activities of a series of alanine scanning mutants. Our study underscores the structural understanding of this protein family and paves the way for their modification to improve the production of neutral lipids.     

A Novel Virulence Strategy for Pseudomonas aeruginosa Mediated by an Autotransporter with Arginine-Specific Aminopeptidase Activity

The opportunistic human pathogen, Pseudomonas aeruginosa, is a major cause of infections in chronic wounds, burns and the lungs of cystic fibrosis patients. The P. aeruginosa genome encodes at least three proteins exhibiting the characteristic three domain structure of autotransporters, but much remains to be understood about the functions of these three proteins and their role in pathogenicity. Autotransporters are the largest family of secreted proteins in Gram-negative bacteria, and those characterised are virulence factors. Here, we demonstrate that the PA0328 autotransporter is a cell-surface tethered, arginine-specific aminopeptidase, and have defined its active site by site directed mutagenesis. Hence, we have assigned PA0328 with the name AaaA, for arginine-specific autotransporter of P. aeruginosa. We show that AaaA provides a fitness advantage in environments where the sole source of nitrogen is peptides with an aminoterminal arginine, and that this could be important for establishing an infection, as the lack of AaaA led to attenuation in a mouse chronic wound infection which correlated with lower levels of the cytokines TNFα, IL-1α, KC and COX-2. Consequently AaaA is an important virulence factor playing a significant role in the successful establishment of P. aeruginosa infections.     

Generation of mice with a conditional allele for G6pc

Glucose‐6‐phosphatase‐α (G6Pase‐α or G6PC) catalyzes the hydrolysis of glucose‐6‐phosphate to glucose and is a key enzyme in interprandial glucose homeostasis. Mutations in the human G6PC gene, expressed primarily in the liver, kidney, and intestine, cause glycogen storage disease Type Ia (GSD‐Ia), an autosomal recessive disorder characterized by a disturbed glucose homeostasis. For better understanding of the roles of G6Pase‐α in different tissues and in pathological conditions, we have generated mice harboring a conditional null allele for G6pc by flanking Exon 3 of the G6pc gene with loxP sites. We confirmed the null phenotype by using the EIIa‐Cre transgenic approach to generate mice lacking Exon 3 of the G6pc gene. The resulting homozygous Cre‐recombined null mice manifest a phenotype mimicking G6Pase‐α‐deficient mice and human GSD‐Ia patients. This G6pc conditional null allele will be valuable to examine the consequence of tissue‐specific G6Pase‐α deficiency and the mechanisms of long‐term complications in GSD‐Ia. genesis 47:590–594, 2009. Published 2009 Wiley‐Liss, Inc.     

A Combinatorial Approach for the Design of Complementarity-determining Region-derived Peptidomimetics with in Vitro Anti-tumoral Activity

The great success of therapeutic monoclonal antibodies has fueled research toward mimicry of their binding sites and the development of new strategies for peptide-based mimetics production. Here, we describe a new combinatorial approach for the production of peptidomimetics using the complementarity-determining regions (CDRs) from gastrin17 (pyroEGPWLEEEEEAYGWMDF-NH₂) antibodies as starting material for cyclic peptide synthesis in a microarray format. Gastrin17 is a trophic factor in gastrointestinal tumors, including pancreatic cancer, which makes it an interesting target for development of therapeutic antibodies. Screening of microarrays containing bicyclic peptidomimetics identified a high number of gastrin binders. A strong correlation was observed between gastrin binding and overall charge of the peptidomimetic. Most of the best gastrin binders proceeded from CDRs containing charged residues. In contrast, CDRs from high affinity antibodies containing mostly neutral residues failed to yield good binders. Our experiments revealed essential differences in the mode of antigen binding between CDR-derived peptidomimetics (K_d values in micromolar range) and the parental monoclonal antibodies (K_d values in nanomolar range). However, chemically derived peptidomimetics from gastrin binders were very effective in gastrin neutralization studies using cell-based assays, yielding a neutralizing activity in pancreatic tumoral cell lines comparable with that of gastrin-specific monoclonal antibodies. These data support the use of combinatorial CDR-peptide microarrays as a tool for the development of a new generation of chemically synthesized cyclic peptidomimetics with functional activity.