Mycobacteriophage endolysins: diverse and modular enzymes with multiple catalytic activities

The mycobacterial cell wall presents significant challenges to mycobacteriophages--viruses that infect mycobacterial hosts--because of its unusual structure containing a mycolic acid-rich mycobacterial outer membrane attached to an arabinogalactan layer that is in turn linked to the peptidoglycan. Although little is known about how mycobacteriophages circumvent these barriers during the process of infection, destroying it for lysis at the end of their lytic cycles requires an unusual set of functions. These include Lysin B proteins that cleave the linkage of mycolic acids to the arabinogalactan layer, chaperones required for endolysin delivery to peptidoglycan, holins that regulate lysis timing, and the endolysins (Lysin As) that hydrolyze peptidoglycan. Because mycobacterial peptidoglycan contains atypical features including 3→3 interpeptide linkages, it is not surprising that the mycobacteriophage endolysins also have non-canonical features. We present here a bioinformatic dissection of these lysins and show that they are highly diverse and extensively modular, with an impressive number of domain organizations. Most contain three domains with a novel N-terminal predicted peptidase, a centrally located amidase, muramidase, or transglycosylase, and a C-terminal putative cell wall binding domain.     

Use of high-affinity cell wall-binding domains of bacteriophage endolysins for immobilization and separation of bacterial cells

Immobilization and magnetic separation for specific enrichment of microbial cells, such as the pathogen Listeria monocytogenes, depends on the availability of suitable affinity molecules. We report here a novel concept for the immobilization and separation of bacterial cells by replacing antibodies with cell wall-binding domains (CBDs) of bacteriophage-encoded peptidoglycan hydrolases (endolysins). These polypeptide modules very specifically recognize and bind to ligands on the gram-positive cell wall with high affinity. With paramagnetic beads coated with recombinant Listeria phage endolysin-derived CBD molecules, more than 90% of the viable L. monocytogenes cells could be immobilized and recovered from diluted suspensions within 20 to 40 min. Recovery rates were similar for different species and serovars of Listeria and were not affected by the presence of other microorganisms. The CBD-based magnetic separation (CBD-MS) procedure was evaluated for capture and detection of L. monocytogenes from artificially and naturally contaminated food samples. The CBD separation method was shown to be superior to the established standard procedures; it required less time (48 h versus 96 h) and was the more sensitive method. Furthermore, the generalizability of the CBD-MS approach was demonstrated by using specific phage-encoded CBDs specifically recognizing Bacillus cereus and Clostridium perfringens cells, respectively. Altogether, CBD polypeptides represent novel and innovative tools for the binding and capture of bacterial cells, with many possible applications in microbiology and diagnostics.     

Bacteriophage endolysins: applications for food safety

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On the repair mechanism responsible for multiplicity reactivation in bacteriophage T4

Summary Multiplicity reactivation has been studied using two T4 phages carrying a large number, twenty six, of genetic markers covering the whole viral genome. It has been found that in the progeny of irradiated phages the observed increase of recombination frequencies is not limited to the vicinity of radiation-induced lethal lesions, it can occur all along the phage genome. Most of the single bursts produced by multiplicity reactivation contain phages of entirely parental genotype. This fact may reveal the existence of repair by recombination between replication products of a single viral genome.     

What makes the bacteriophage λ Red system useful for genetic engineering: molecular mechanism and biological function

Recent studies have generated interest in the use of the homologous recombination system of bacteriophage lambda for genetic engineering. The system, called Red, consists primarily of three proteins: lambda exonuclease, which processively digests the 5'-ended strand of a dsDNA end; beta protein, which binds to ssDNA and promotes strand annealing; and gamma protein, which binds to the bacterial RecBCD enzyme and inhibits its activities. These proteins induce a 'hyper-rec' state in Escherichia coli and other bacteria, in which recombination events between DNA species with as little as 40 bp of shared sequence occur at high frequency. Red-mediated recombination in the hyper-rec bacterium proceeds via a number of different pathways, and with the involvement of different sets of bacterial proteins, depending in part on the nature of the recombining DNA species. The role of high-frequency double-strand break repair/recombination in the life cycle of the lambdoid phages is discussed.     

Domain shuffling and module engineering of Listeria phage endolysins for enhanced lytic activity and binding affinity

Bacteriophage endolysins are peptidoglycan hydrolases employed by the virus to lyse the host at the end of its multiplication phase. They have found many uses in biotechnology; not only as antimicrobials, but also for the development of novel diagnostic tools for rapid detection of pathogenic bacteria. These enzymes generally show a modular organization, consisting of N‐terminal enzymatically active domains (EADs) and C‐terminal cell wall‐binding domains (CBDs) which specifically target the enzymes to their substrate in the bacterial cell envelope. In this work, we used individual functional modules of Listeria phage endolysins to create fusion proteins with novel and optimized properties for labelling and lysis of Listeria cells. Chimaeras consisting of individual EAD and CBD modules from PlyPSA and Ply118 endolysins with different binding specificity and catalytic activity showed swapped properties. EAD118–CBDPSA fusion proteins exhibited up to threefold higher lytic activity than the parental endolysins. Recombineering different CBD domains targeting various Listeria cell surfaces into novel heterologous tandem proteins provided them with extended recognition and binding properties, as demonstrated by fluorescent GFP‐tagged CBD fusions. It was also possible to combine the binding specificities of different single CBDs in heterologous tandem CBD constructs such as CBD500‐P35 and CBDP35‐500, which were then able to recognize the majority of Listeria strains. Duplication of CBD500 increased the equilibrium cell wall binding affinity by approximately 50‐fold, and the enzyme featuring tandem CBD modules showed increased activity at higher ionic strength. Our results demonstrate that modular engineering of endolysins is a powerful approach for the rational design and optimization of desired functional properties of these proteins.     

On the catalytic mechanism of human ATP citrate lyase

ATP citrate lyase (ACL) catalyzes an ATP-dependent biosynthetic reaction which produces acetyl-coenzyme A and oxaloacetate from citrate and coenzyme A (CoA). Studies were performed with recombinant human ACL to ascertain the nature of the catalytic phosphorylation that initiates the ACL reaction and the identity of the active site residues involved. Inactivation of ACL by treatment with diethylpyrocarbonate suggested the catalytic role of an active site histidine (i.e., His760), which was proposed to form a phosphohistidine species during catalysis. The pH-dependence of the pre-steady-state phosphorylation of ACL with [γ-(33)P]-ATP revealed an ionizable group with a pK(a) value of ~7.5, which must be unprotonated for the catalytic phosphorylation of ACL to occur. Mutagenesis of His760 to an alanine results in inactivation of the biosynthetic reaction of ACL, in good agreement with the involvement of a catalytic histidine. The nature of the formation of the phospho-ACL was further investigated by positional isotope exchange using [γ-(18)O(4)]-ATP. The β,γ-bridge to nonbridge positional isotope exchange rate of [γ-(18)O(4)]-ATP achieved its maximal rate of 14 s(-1) in the absence of citrate and CoA. This rate decreased to 5 s(-1) when citrate was added, and was found to be 10 s(-1) when both citrate and CoA were present. The rapid positional isotope exchange rates indicated the presence of one or more catalytically relevant, highly reversible phosphorylated intermediates. Steady-state measurements in the absence of citrate and CoA showed that MgADP was produced by both wild type and H760A forms of ACL, with rates at three magnitudes lower than that of k(cat) for the full biosynthetic reaction. The ATPase activity of ACL, along with the small yet significant positional isotope exchange rate observed in H760A mutant ACL (~150 fold less than wild type), collectively suggested the presence of a second, albeit unproductive, phosphoryl transfer in ACL. Mathematical analysis and computational simulation suggested that the desorption of MgADP at a rate of ~7 s(-1) was the rate-limiting step in the biosynthesis of AcCoA and oxaloacetate.     

Opportunities for yeast metabolic engineering: Lessons from synthetic biology

Constant progress in genetic engineering has given rise to a number of promising areas of research that facilitated the expansion of industrial biotechnology. The field of metabolic engineering, which utilizes genetic tools to manipulate microbial metabolism to enhance the production of compounds of interest, has had a particularly strong impact by providing new platforms for chemical production. Recent developments in synthetic biology promise to expand the metabolic engineering toolbox further by creating novel biological components for pathway design. The present review addresses some of the recent advances in synthetic biology and how these have the potential to affect metabolic engineering in the yeast Saccharomyces cerevisiae. While S. cerevisiae for years has been a robust industrial organism and the target of multiple metabolic engineering trials, its potential for synthetic biology has remained relatively unexplored and further research in this field could strongly contribute to industrial biotechnology. This review also addresses are general considerations for pathway design, ranging from individual components to regulatory systems, overall pathway considerations and whole-organism engineering, with an emphasis on potential contributions of synthetic biology to these areas. Some examples of applications for yeast synthetic biology and metabolic engineering are also discussed.     

Lytic activity of recombinant bacteriophage phi11 and phi12 endolysins on whole cells and biofilms of Staphylococcus aureus

The recombinant phi11 endolysin hydrolyzed heat-killed staphylococci as well as staphylococcal biofilms. Cell wall targeting appeared to be a prerequisite for lysis of whole cells, and the combined action of the endopeptidase and amidase domains was necessary for maximum activity. In contrast, the phi12 endolysin was inactive and caused aggregation of the cells.     

A protein engineering study of the role of aspartate 158 in the catalytic mechanism of papain

The controversy concerning the various suggested roles for the side chain of Asp158 in the active site of papain has been clarified by using site-directed mutagenesis. Both wild-type papain and an Asp158 Asn variant were produced in a baculovirus-insect cell expression system, purified to homogeneity from the culture, and characterized kinetically. With CBZ-Phe-Arg-MCA as substrate, the kcat/KM and kcat values obtained for the Asp158Asn papain are 20,000 M-1.s-1 and 34 s-1, respectively, as compared with values of 120,000 M-1.s-1 and 51 s-1 obtained for the wild-type papain. In addition, the pH-(kcat/KM) profile for the Asp158Asn enzyme is shifted relative to that for the wild-type enzyme to lower values by approximately 0.3 pH unit. This shows clearly that Asp158 is not, as previously postulated, an essential catalytic residue. In addition, the pH dependency data are interpreted to indicate that, contrary to earlier suggestions, the negatively charged side chain of Asp158 does not significantly stabilize the active-site thiolate-imidazolium ion pair. However, its presence does influence the pKa's associated with ion-pair formation in a manner compatible with electrostatic considerations.     

On the three-dimensional structure and catalytic mechanism of triose phosphate isomerase

Triose phosphate isomerase is a dimeric enzyme of molecular mass 56 000 which catalyses the interconversion of dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate. The crystal structure of the enzyme from chicken muscle has been determined at a resolution of 2.5 A, and an independent determination of the structure of the yeast enzyme has just been completed at 3 A resolution. The conformation of the polypeptide chain is essentially identical in the two structures, and consists of an inner cylinder of eight strands of parallel beta-pleated sheet, with mostly helical segments connecting each strand. The active site is a pocket containing glutamic acid 165, which is believed to act as a base in the reaction. Crystallographic studies of the binding of DHAP to both the chicken and the yeast enzymes reveal a common mode of binding and suggest a mechanisms for catalysis involving polarization of the substrate carbonyl group.     

Human cytidine deaminase: understanding the catalytic mechanism

In the absence of an experimentally elucidated three-dimensional structure of the human CDA, we built an homology model of this enzyme starting from the crystal structure of its E. coli homologous. Furthermore, we docked in the active site alternatively the substrate, the intermediate or the product. By means of molecular dynamics simulations, we determined the topology of the active site, identifying the amino acids involved in the catalytic mechanism, and outlining the central role played by E67.     

Genetic engineering and accelerated plant improvement: Opportunities and challenges

Plant Cell, Tissue and Organ Culture (PCTOC) -