Ricin-like plant toxins are evolutionarily related to single-chain ribosome-inhibiting proteins from Phytolacca

A comparison has been made of the amino-terminal sequences of a number of ribosome-inhibiting proteins (RIPs) and cytotoxins. These include the monomeric enzymes PAP, PAP-S, PAP-II, and dodecandrin and the enzymatic A chains from the heterodimeric toxins ricin and modeccin. We show that these proteins have all evolved from a single ancestor. A statistical analysis is used to show the likely evolutionary relationship among the proteins. A similar analysis was performed on the amino-terminal sequences of ricin, Ricinus agglutinin, and modeccin B chains. These are galactoside-binding proteins associated with the A-chain enzymes. From the two comparisons we propose a scheme for the development of two major classes of proteins. The RIP and sugar-binding genes probably evolved independently. In some plant lines the genes never fused, although the RIP gene replicated and developed into several proteins expressed at various stages of plant maturation. In another line the RIP gene fused with a sugar binding (B-chain) gene to form the class of heterodimeric toxins. In some species this fused gene appears to have multiplied, one or more of the toxin genes mutating to code for a self-dimerizing agglutinin molecule.     

The N alpha-acetylenkephalin carboxypeptidase activity of N-acetyltyrosine deacetylase from monkey kidney. Purification, characterization and substrate specificity.

N alpha-Acetylenkephalin carboxypeptidase was co-purified with N-acetyltyrosine deacetylase from monkey kidney. Almost 90% of the activity from the homogenate was recovered in a high-speed supernatant without the use of detergents. The crucial steps in the purification were Cibacron Blue F3GA--Sepharose chromatography (involving negative and positive binding sequentially) and metal chelate affinity chromatography. The purified enzyme showed three bands on gel electrophoresis under non-denaturing conditions. All the three bands exhibited both N-acetyltyrosine deacetylase and N-acetylenkephalin carboxypeptidase activity, indicating their co-migration, Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in the presence and absence of 2-mercaptoethanol gave a single protein band of mol.wt. 34 000. The native enzyme was a dimer of mol.wt. 66 000 as observed on Bio-Gel P-300 gel filtration. The carboxypeptidase removed two amino acids from the C-terminal end of either N-acetyl[Met5]- or N-acetyl[Leu5]-enkephalin. Non-acetylated enkephalins were less active as substrates. Peptides with their carboxy end blocked were inactive as substrates. Models suggested for carboxypeptidase A [Hartsuck & Lipscomb (1971) Enzymes 3, 1-56] support the idea that the kidney N-acetylated aromatic amino acid deacetylase or acylase III [Endo (1978) Biochim. Biophys. Acta 523, 207-217] can act as a carboxypeptidase on peptides having hydrophobic amino acids at the C-terminal end.     

Identification of four evolutionarily related G protein-coupled receptors from the malaria mosquito Anopheles gambiae

The mosquito Anopheles gambiae is an important vector for malaria, which is one of the most serious human parasitic diseases in the world, causing up to 2.7 million deaths yearly. To contribute to our understanding of A. gambiae and to the transmission of malaria, we have now cloned four evolutionarily related G protein-coupled receptors (GPCRs) from this mosquito and expressed them in Chinese hamster ovary cells. After screening of a library of thirty-three insect or other invertebrate neuropeptides and eight biogenic amines, we could identify (de-orphanize) three of these GPCRs as: an adipokinetic hormone (AKH) receptor (EC₅₀ for A. gambiae AKH, 3 × 10⁻⁹ M), a corazonin receptor (EC₅₀ for A. gambiae corazonin, 4 × 10⁻⁹ M), and a crustacean cardioactive peptide (CCAP) receptor (EC₅₀ for A. gambiae CCAP, 1 × 10⁻⁹ M). The fourth GPCR remained an orphan, although its close evolutionary relationship to the A. gambiae and other insect AKH receptors suggested that it is a receptor for an AKH-like peptide. This is the first published report on evolutionarily related AKH, corazonin, and CCAP receptors in mosquitoes.     

Bacterial N-succinyl-L-diaminopimelic acid desuccinylase. Purification, partial characterization, and substrate specificity

The enzyme N-succinyl-L-diaminopimelic acid desuccinylase from Escherichia coli has been purified 7,100-fold to apparent homogeneity. The enzyme is part of the diaminopimelic acid-lysine pathway in bacteria and catalyzes the hydrolysis of N-succinyl-L-diaminopimelic acid to produce L-diaminopimelic acid and succinate. The enzyme exists as a mixture of dimeric and tetrameric species of identical subunits of molecular weight approximately 40,000. Activity was completely abolished following dialysis of the enzyme against metal chelators. Cobalt(II) and zinc were effective in restoring the activity. The apparent affinities of the apoenzyme for cobalt and zinc were similar (Kd values near 1 microM) and the cobalt enzyme was 2.2-fold more active than the zinc enzyme. The Km and turnover number for the hydrolysis of the natural substrate, N-succinyl-L-diaminopimelic acid, were 0.4 mM and 16,000 min-1, respectively. The substrate specificity of the enzyme was defined by preparing a number of substrate analogues that systematically lack the various functional groups present in the molecule. These studies show that the enzyme is highly specific for the natural substrate. These properties of N-succinyl-L-diaminopimelic acid desuccinylase and the fact that the enzyme is essential for bacterial growth make it an ideal target for the development of inhibitors with potential antibacterial activity.     

Crystallization and preliminary crystallographic analysis of carboxypeptidase G2 from Pseudomonas sp. strain RS-16

Carboxypeptidase G2, a zinc metalloenzyme isolated from Pseudomonas sp. strain RS-16, which catalyses the hydrolytic cleavage of reduced and non-reduced folates to pteroates and L-glutamate, has been crystallized from polyethylene glycol (average Mr 4000) by vapour diffusion. The crystal symmetry is monoclinic C2, with unit cell dimensions a = 206 A, b = 82 A, c = 116 A and beta = 118 degrees. The molecular mass and volume of the unit cell suggest that there are two dimers of the enzyme in the asymmetric unit. The crystals diffract to at least 3.0 A and are suitable for X-ray structure analysis.     

Characterization of the carboxypeptidase N secreted by Hep G2 cells

Human hepatoma (Hep G2) cells secrete nanogram quantities of carboxypeptidase enzymes which are capable of hydrolyzing COOH-terminal lysine and arginine residues. A carboxypeptidase with a neutral pH optimum (greater than pH 7.0) was partially purified from the conditioned medium and compared with pure plasma carboxypeptidase N. The two enzymes behaved in a similar manner on gel filtration (apparent Mr = 280,000), DE52 ion exchange chromatography, and concanavalin A-affinity chromatography and were indistinguishable enzymatically and immunologically. Immunoblots of the Hep G2 and plasma carboxypeptidase N before and following deglycosylation with peptide-N4-[N-acetyl-beta-glucosaminyl]asparagine amidase F revealed a similar, if not identical, multimeric structure. A second carboxypeptidase with a lower molecular weight and a pH optimum of 5.0 was also detected in the Hep G2 medium.     

Phage endolysins are adapted to specific hosts and are evolutionarily dynamic

Endolysins are produced by (bacterio)phages to rapidly degrade the bacterial cell wall and release new viral particles. Despite sharing a common function, endolysins present in phages that infect a specific bacterial species can be highly diverse and vary in types, number, and organization of their catalytic and cell wall binding domains. While much is now known about the biochemistry of phage endolysins, far less is known about the implication of their diversity on phage–host adaptation and evolution. Using CRISPR-Cas9 genome editing, we could genetically exchange a subset of different endolysin genes into distinct lactococcal phage genomes. Regardless of the type and biochemical properties of these endolysins, fitness costs associated to their genetic exchange were marginal if both recipient and donor phages were infecting the same bacterial strain, but gradually increased when taking place between phage that infect different strains or bacterial species. From an evolutionary perspective, we observed that endolysins could be naturally exchanged by homologous recombination between phages coinfecting a same bacterial strain. Furthermore, phage endolysins could adapt to their new phage/host environment by acquiring adaptative mutations. These observations highlight the remarkable ability of phage lytic systems to recombine and adapt and, therefore, explain their large diversity and mosaicism. It also indicates that evolution should be considered to act on functional modules rather than on bacteriophages themselves. Furthermore, the extensive degree of evolvability observed for phage endolysins offers new perspectives for their engineering as antimicrobial agents.

Endolysins are produced by bacteriophages to degrade the host cell wall and release new particles, but the implications of their diversity on phage-host adaptation and evolution is unknown. This study uses CRISPR-Cas9 genome editing to reveal novel insights into bacteriophage endolysin diversity and phage-bacteria interactions as well as into endolysin adaptation towards a new bacterial host.     

Spinach glycolate oxidase and yeast flavocytochrome b2 are structurally homologous and evolutionarily related enzymes with distinctly different function and flavin mononucleotide binding

A comparison of the three-dimensional structures of the flavin mononucleotide (FMN)-dependent enzymes glycolate oxidase, flavocytochrome b2, and trimethylamine dehydrogenase is presented. Their flavin-binding domains all have the same structural motif, the 8-fold beta/alpha-barrel domain, which is also present in a large number of other enzymes. FMN is bound in a similar fashion in all three enzymes. The binding site is at the carboxyl-terminal end of the eight beta-strands of the barrel where the active site is invariably found in this type of domain structure. The similarity of the structures of glycolate oxidase and flavocytochrome b2 extends to the loop regions and even outside the beta/alpha-barrels with a root mean square deviation of 0.93 A for 311 superimposed C alpha-atoms and with a sequence identity of 37%. A detailed analysis of their active sites shows, however, that the orientation of FMN is significantly different in the two structures due to different conformations of residues in the end of strand one. Thus, in flavocytochrome b2 a hydrogen bond is formed between the FMN N-5 position and the main chain amide of Ala-198, while in glycolate oxidase, the ring system is tilted away from the strand, creating a pocket on the re-side of the FMN ring where a water molecule is bound. Model building shows that this site could accommodate the hydroperoxide moiety of a FMN-4a-hydroperoxide intermediate. Thus, in the course of evolution, a few mutations in, and close to, the active sites have fine tuned these enzymes to exert their specific functions as an oxidase or transferase, respectively.     

Isolation, purification, and characterization of a new enzyme from Pseudomonas sp. M-27, carboxypeptidase G3.

A new type of carboxypeptidase was found in a strain of Pseudomonas sp. M-27 isolated from soil. The cell-free extract, solubilized by colistin sulfate, was purified to homogeneity. This enzyme had a single peak with a molecular weight of 60,000 on a calibrated Superdex column and consisted of four subunits of identical molecular weights (M(r): 15,000). The enzyme hydrolyzed predominantly acidic peptides and N-acyl amino acids with Glu or Asp in the C-termini. This enzyme was not strongly affected by thiol enzyme inhibitors (PCMB, iodoacetic acid) or serine protease inhibitors (DFP, PMSF), but was inhibited by metal chelators. The enzyme resembles carboxypeptidase G1 or G2 in its glutamate-releasing activity. However, it acts not only on the L-form but also on the D-form of acidic amino acids and shows affinity for the long-chain fatty acyl group but not the benzoyl group. Thus, as this enzyme differs from carboxypeptidase G1 or G2, it was named carboxypeptidase G3.     

Human myeloperoxidase and thyroid peroxidase, two enzymes with separate and distinct physiological functions, are evolutionarily related members of the same gene family

Human myeloperoxidase and human thyroid peroxidase nucleotide and amino acid sequences were compared. The global similarities of the nucleotide and amino acid sequences are 46% and 44%, respectively. These similarities are most evident within the coding sequence, especially that encoding the myeloperoxidase functional subunits. These results clearly indicate that myeloperoxidase and thyroid peroxidase are members of the same gene family and diverged from a common ancestral gene. The residues at 416 in myeloperoxidase and 407 in thyroid peroxidase were estimated as possible candidates for the proximal histidine residues that link to the iron centers of the enzymes. The primary structures around these histidine residues were compared with those of other known peroxidases. The similarity in this region between the two animal peroxidases (amino acid 396-418 in thyroid peroxidase and 405-427 in myeloperoxidase) is 74%; however, those between the animal peroxidases and other yeast and plant peroxidases are not significantly high, although several conserved features have been observed. The possible location of the distal histidine residues in myeloperoxidase and thyroid peroxidase amino acid sequences are also discussed.     

Production of cloned carboxypeptidase G2 by Escherichia coli: Genetic and environmental considerations

Summary The optimum production of cloned carboxypeptidase G₂ from plasmid pNM21 byEscherichia coli was found to be strongly strain- and temperature-dependent. The superior host was strain RV308 and the preferred growth temperature 28°C. Copy number, which decreased during exponential growth of all strains examined, did not relate in these studies to the level of enzyme production: the strain with the highest enzyme yield also having the lowest overall copy number.     

Epstein-Barr virus microRNAs are evolutionarily conserved and differentially expressed

The pathogenic lymphocryptovirus Epstein-Barr virus (EBV) is shown to express at least 17 distinct microRNAs (miRNAs) in latently infected cells. These are arranged in two clusters: 14 miRNAs are located in the introns of the viral BART gene while three are located adjacent to BHRF1. The BART miRNAs are expressed at high levels in latently infected epithelial cells and at lower, albeit detectable, levels in B cells. In contrast to the tissue-specific expression pattern of the BART miRNAs, the BHRF1 miRNAs are found at high levels in B cells undergoing stage III latency but are essentially undetectable in B cells or epithelial cells undergoing stage I or II latency. Induction of lytic EBV replication was found to enhance the expression of many, but not all, of these viral miRNAs. Rhesus lymphocryptovirus, which is separated from EBV by > or =13 million years of evolution, expresses at least 16 distinct miRNAs, seven of which are closely related to EBV miRNAs. Thus, lymphocryptovirus miRNAs are under positive selection and are likely to play important roles in the viral life cycle. Moreover, the differential regulation of EBV miRNA expression implies distinct roles during infection of different human tissues.     

Evidence that Hoxa expression domains are evolutionarily transposed in spinal ganglia, and are established by forward spreading in paraxial mesoderm.

Transposition of anatomical structures along the anteroposterior axis has been a commonly used mechanism for changing body proportions during the course of evolutionary time. Earlier work (Gaunt, S.J., 1994. Conservation in the Hox code during morphological evolution. Int. J. Dev. Biol. 38, 549-552; Burke, A.C., Nelson, C.E., Morgan, B.A., Tabin, C., 1995. Hox genes and the evolution of vertebrate axial morphology. Development 121, 333-346) showed how transposition in mesodermal derivatives (vertebrae) could be attributed to transposition in the expression of Hox genes along the axial series of somites. We now show how transposition in the segmental arrangement of the spinal nerves can also be correlated with shifts in the expression domains of Hox genes. Specifically, we show how the expression domains of Hoxa-7, a-9 and a-10 in spinal ganglia correspond similarly in both mouse and chick with the positions of the brachial and lumbosacral plexuses, and that this is true even though the brachial plexus of chick is shifted posteriorly, relative to mouse, by seven segmental units. In spite of these marked species differences in the boundaries of Hoxa-7 expression, cis regulatory elements located up to 5 kb upstream of the chick Hoxa-7 gene showed much functional and structural conservation with those described in the mouse (Puschel, A.W., Balling, R., Gruss, P., 1991. Separate elements cause lineage restriction and specify boundaries of Hox-1.1 expression. Development 112, 279-287; Knittel, T., Kessel, M., Kim, M.H., Gruss, P., 1995. A conserved enhancer of the human and murine Hoxa-7 gene specifies the anterior boundary of expression during embryonal development. Development 121, 1077-1088). We also show that chick Hoxa-7 and a-10 expression domains spread forward into regions of somites that are initially negative for the expression of these genes. We discuss this as evidence that Hox expression in paraxial mesoderm spreads forward, as earlier found for neurectoderm and lateral plate mesoderm, in a process that occurs independently of cell movement.     

Expression of the Pseudomonas gene coding for carboxypeptidase G2 in Saccharomyces cerevisiae

The Pseudomonas gene coding for carboxypeptidase G2 was introduced into Saccharomyces cerevisiae on an Escherichia coli/yeast shuttle vector pROG5. The level of enzyme activity obtained was independent of the orientation of the gene within the pBR322-derived tetracycline resistance gene of the vector, indicating that expression can occur from a Pseudomonas promoter in yeast.