Norbert Sträter

Cellular function and molecular structure of ecto-nucleotidases

Ecto-nucleotidases play a pivotal role in purinergic signal transmission. They hydrolyze extracellular nucleotides and thus can control their availability at purinergic P2 receptors. They generate extracellular nucleosides for cellular reuptake and salvage via nucleoside transporters of the plasma membrane. The extracellular adenosine formed acts as an agonist of purinergic P1 receptors. They also can produce and hydrolyze extracellular inorganic pyrophosphate that is of major relevance in the control of bone mineralization. This review discusses and compares four major groups of ecto-nucleotidases: the ecto-nucleoside triphosphate diphosphohydrolases, ecto-5′-nucleotidase, ecto-nucleotide pyrophosphatase/phosphodiesterases, and alkaline phosphatases. Only recently and based on crystal structures, detailed information regarding the spatial structures and catalytic mechanisms has become available for members of these four ecto-nucleotidase families. This permits detailed predictions of their catalytic mechanisms and a comparison between the individual enzyme groups. The review focuses on the principal biochemical, cell biological, catalytic, and structural properties of the enzymes and provides brief reference to tissue distribution, and physiological and pathophysiological functions.

X‐ray structure of aminopeptidase A from Escherichia coli and a model for the nucleoprotein complex in Xer site‐specific recombination

The structure of aminopeptidase A (PepA), which functions as a DNA‐binding protein in Xer site‐specific recombination and in transcriptional control of the carAB operon in Escherichia coli, has been determined at 2.5 Å resolution. In Xer recombination at cer, PepA and the arginine repressor (ArgR) serve as accessory proteins, ensuring that recombination is exclusively intramolecular. In contrast, PepA homologues from other species have no known DNA‐binding activity and are not implicated in transcriptional regulation or control of site‐specific recombination. PepA comprises two domains, which have similar folds to the two domains of bovine lens leucine aminopeptidase (LAP). However, the N‐terminal domain of PepA, which probably plays a significant role in DNA binding, is rotated by 19° compared with its position in LAP. PepA is a homohexamer of 32 symmetry. A groove that runs from one trimer face across the 2‐fold molecular axis to the other trimer face is proposed to be the DNA‐binding site. Molecular modelling supports a structure of the Xer complex in which PepA, ArgR and a second PepA molecule are sandwiched along their 3‐fold molecular axes, and the accessory sequences of the two recombination sites wrap around the accessory proteins as a right‐handed superhelix such that three negative supercoils are trapped.

Ecto-5'-nucleotidase: Structure function relationships.

Ecto-5’-nucleotidase (ecto-5’-NT) is attached via a GPI anchor to the extracellular membrane, where it hydrolyses AMP to adenosine and phosphate. Related 5’-nucleotidases exist in bacteria, where they are exported into the periplasmic space. X-ray structures of the 5’-nucleotidase from E. coli showed that the enzyme consists of two domains. The N-terminal domain coordinates two catalytic divalent metal ions, whereas the C-terminal domain provides the substrate specificity pocket for the nucleotides. Thus, the substrate binds at the interface of the two domains. Here, the currently available structural information on ecto-5’NT is reviewed in relation to the catalytic properties and enzyme function.

Crystal structure of the plasmid maintenance system epsilon/zeta: functional mechanism of toxin zeta and inactivation by epsilon 2 zeta 2 complex formation.

Programmed cell death in prokaryotes is frequently found as postsegregational killing. It relies on antitoxin/toxin systems that secure stable inheritance of low and medium copy number plasmids during cell division and kill cells that have lost the plasmid. The broad-host-range, low-copy-number plasmid pSM19035 from Streptococcus pyogenes carries the genes encoding the antitoxin/toxin system ɛ/ζ and antibiotic resistance proteins, among others. The crystal structure of the biologically nontoxic ɛ2ζ2 protein complex at a 1.95-Å resolution and site-directed mutagenesis showed that free ζ acts as phosphotransferase by using ATP/GTP. In ɛ2ζ2, the toxin ζ is inactivated because the N-terminal helix of the antitoxin ɛ blocks the ATP/GTP-binding site. To our knowledge, this is the first prokaryotic postsegregational killing system that has been entirely structurally characterized.

The X-ray Crystal Structure of Human β-Hexosaminidase B Provides New Insights into Sandhoff Disease

Human lysosomal beta-hexosaminidases are dimeric enzymes composed of alpha and beta-chains, encoded by the genes HEXA and HEXB. They occur in three isoforms, the homodimeric hexosaminidases B (betabeta) and S (alphaalpha), and the heterodimeric hexosaminidase A (alphabeta), where dimerization is required for catalytic activity. Allelic variations in the HEXA and HEXB genes cause the fatal inborn errors of metabolism Tay-Sachs disease and Sandhoff disease, respectively. Here, we present the crystal structure of a complex of human beta-hexosaminidase B with a transition state analogue inhibitor at 2.3A resolution (pdb 1o7a). On the basis of this structure and previous studies on related enzymes, a retaining double-displacement mechanism for glycosyl hydrolysis by beta-hexosaminidase B is proposed. In the dimer structure, which is derived from an analysis of crystal packing, most of the mutations causing late-onset Sandhoff disease reside near the dimer interface and are proposed to interfere with correct dimer formation. The structure reported here is a valid template also for the dimeric structures of beta-hexosaminidase A and S.

Insect‐Derived Proline‐Rich Antimicrobial Peptides Kill Bacteria by Inhibiting Bacterial Protein Translation at the 70 S Ribosome

Proline-rich antimicrobial peptides (PrAMPs) have been investigated and optimized by several research groups and companies as promising lead compounds to treat systemic infections caused by Gram-negative bacteria. PrAMPs, such as apidaecins and oncocins, enter the bacteria and kill them apparently through inhibition of specific targets without a lytic effect on the membranes. Both apidaecins and oncocins were shown to bind with nanomolar dissociation constants to the 70S ribosome. In apidaecins, at least the two C-terminal residues (Arg17 and Leu18) interact strongly with the 70S ribosome, whereas residues Lys3, Tyr6, Leu7, and Arg11 are the major interaction sites in oncocins. Oncocins inhibited protein biosynthesis very efficiently in vitro with half maximal inhibitory concentrations (IC50 values) of 150 to 240 nmol L(-1). The chaperone DnaK is most likely not the main target of PrAMPs but it binds them with lower affinity.

STRUCTURAL RELATIONSHIP BETWEEN THE MAMMALIAN FE(III)-FE(II) AND THE FE(III)-ZN(II) PLANT PURPLE ACID PHOSPHATASES

The primary structure of uteroferrin (Uf), a 35 kDa monomeric mammalian purple acid phosphatase (PAP) containing a Fe(III)‐Fe(II) center, has been compared with the sequence of the homodimeric 111 kDa Fe(III)‐Zn(II) kidney bean purple acid phosphatase (KBPAP). The alignment suggests that the amino acid residues ligating the dimetal center are identical in Uf and KBPAP, although the geometry of the coordination sphere might slightly differ. Secondary structure predictions indicate that Uf contains two βαβαβ motifs thus resembling the folding topology of the plant enzyme. Guided by the recently determined X‐ray structure of KBPAP a tentative model for the mammalian PAP can be constructed.

Api88 Is a Novel Antibacterial Designer Peptide To Treat Systemic Infections with Multidrug-Resistant Gram-Negative Pathogens

The emergence of multiple-drug-resistant (MDR) bacterial pathogens in hospitals (nosocomial infections) presents a global threat of growing importance, especially for Gram-negative bacteria with extended spectrum β-lactamase (ESBL) or the novel New Delhi metallo-β-lactamase 1 (NDM-1) resistance. Starting from the antibacterial peptide apidaecin 1b, we have optimized the sequence to treat systemic infections with the most threatening human pathogens, such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. The lead compound Api88 enters bacteria without lytic effects at the membrane and inhibits chaperone DnaK at the substrate binding domain with a K(D) of 5 μmol/L. The Api88-DnaK crystal structure revealed that Api88 binds with a seven residue long sequence (PVYIPRP), in two different modes. Mice did not show any sign of toxicity when Api88 was injected four times intraperitoneally at a dose of 40 mg/kg body weight (BW) within 24 h, whereas three injections of 1.25 mg/kg BW and 5 mg/kg BW were sufficient to rescue all animals in lethal sepsis models using pathogenic E. coli strains ATCC 25922 and Neumann, respectively. Radioactive labeling showed that Api88 enters all organs investigated including the brain and is cleared through both the liver and kidneys at similar rates. In conclusion, Api88 is a novel, highly promising, 18-residue peptide lead compound with favorable in vitro and in vivo properties including a promising safety margin.

Rational Design of Oncocin Derivatives with Superior Protease Stabilities and Antibacterial Activities Based on the High-Resolution Structure of the Oncocin-DnaK Complex.

Despite the success story of antibiotics, which began nearly a hundred years ago, bacterial infections are still a major cause of death worldwide. The emergence of multiple-drug-resistant (MDR) bacterial pathogens in hospitals (nosocomial infections) presents a global problem of growing importance, with an estimated annual death toll of 50 000 in the EU and 60 000 in the USA. More recently, MDR bacteria have also caused severe community-acquired infections, indicating that we will soon face bacterial strains with the ability to overcome existing antibiotic treatments, and which will therefore represent a severe global threat. Agents responsible for important resistance mechanisms in Gram-negative bacteria include extended spectrum b-lactamases (ESBLs) in Enterobacteriaceae (e.g. , E. coli, K. pneumoniae, and Enterobacter cloacae) or broad-range b-lactamases (e.g. , KPC in Klebsiella pneumoniae or metallo-b-lactamases in Pseudomonas aeruginosa). MDR Acinetobacter baumannii, associated with invasive infections such as pneumonia, meningitis, and bacteremia, has been found to be responsible for outbreaks in intensive care units, including “panresistant” A. baumannii clones susceptible only to polymyxin. To provide effective future treatment options, novel antimicrobial drug classes with novel modes of action are urgently needed. Inducible, gene-encoded antimicrobial peptides (AMPs) represent such a promising alternative, having been selected and optimized by evolution over millions of years. Although AMPs that kill bacteria by lytic effects on the membrane are often toxic to human cells at higher doses, the class of small prolinerich AMPs (PR-AMPs) expressed in mammals and insects has attracted considerable interest. These peptides specifically target intracellular components in Gram-negative bacteria with no indication of any resulting toxic side effects. Despite their favorable antibacterial spectrum against Enterobacteriaceae and nonfermenting species (e.g. , A. baumannii and P. aeruginosa), there are multiple obstacles to be overcome in their further development for therapeutic consideration. We have recently used rational design to optimize the 19-residue-long PR-AMP oncocin (VDKPPYLPRPRPPRRIYNR-NH2) as a potential means of countering the five human pathogens discussed. Substitution of Arg15 and Arg19 by ornithine drastically improved the half-life in mouse serum. Mechanistically, oncocin freely penetrates the bacterial membrane and distributes homogenously within E. coli cells. Here we report its further optimization, based on a positional Ala-scan to deduce residues critical to its antibacterial activity and the crystal structure of an oncocin-DnaK (ligand–target) complex. The new lead compounds were highly resistant against serum (t1=2 8 h) and E. coli proteases (t1=2 >10 h). The mode of action assumed for oncocin and all other PRAMPs has not been worked out in detail, but most likely consists of at least three steps: passive penetration of the bacterial outer membrane, active transport from the periplasmic space into the cytoplasm, and inhibition of DnaK and maybe other targets. 12] A lead optimization strategy therefore has to consider all aspects and cannot focus only on the target binding. We first identified residues crucial for the antibacterial activity of oncocin by determining the minimal inhibitory concentrations (MICs) and inhibition zones for all 19 peptides resulting from a positional Ala-scan (see Figure S1 in the Supporting Information). Substitution of residues 1, 2, 4, 5, 10, and 12–19 reduced the antibacterial activity slightly, whereas substitutions at positions 3, 6–9, and 11 abolished it almost completely. Although the target of oncocin has not yet been identified, the high sequence homology to other insect-derived PR-AMPs, especially pyrrhocoricin, suggests that it might be the bacterial chaperone DnaK. Full-length DnaK was therefore expressed in E. coli and purified in order to study oncocin binding by fluorescence polarization. The binding constants for oncocin and its analogue O2, with a 5(6)-carboxyfluorescein unit at the N terminus, were 1.0 0.2 mm and 4.0 1.0 mm, respectively, whereas all-d oncocin did not bind (Table 1, Figures S2 and S3). These values correspond to binding constants reported for other DnaK-binding sequences, ranging from 0.1 to 10 mm. Cocrystallization of oncocin O2 with the substrate binding domain of DnaK (residues 389 to 607), demonstrated that oncocin residues 4 to 10 (PPYLPR) bound to the peptide binding site of DnaK, whereas the remaining terminal residues of the peptide were flexible (Figures 1 and S4–S8). Interestingly, this sequence stretch matched the residues identified by the Ala-scan as crucial for antibacterial activity. The antibacterial activity of oncocin thus mostly depends on its DnaK binding site, although the activity is also abolished by shortening the sequence, either from the N or the C terminus. Such inactivation, which can occur through the action of proteases either in the bacteria or in blood, should be minimized for systemic applications. Because the peptide O2 is rel[a] D. Knappe, M. Zahn, Prof. Dr. N. Str ter, Prof. Dr. R. Hoffmann Institut f r Bioanalytische Chemie Biotechnologisch-Biomedizinisches Zentrum Fakult t f r Chemie und Mineralogie, Universit t Leipzig Deutscher Platz 5, 04103 Leipzig (Germany) Fax: (+ 49) 341-97-31339 E-mail : hoffmann@chemie.uni-leipzig.de [b] U. Sauer, Dr. G. Schiffer AiCuris GmbH & Co KG Friedrich-Ebert-Strasse 475, Building 302, 42117 Wuppertal (Germany) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201000792.

X-ray structure of acarbose bound to amylomaltase from Thermus aquaticus Implications for the synthesis of large cyclic glucans

As a member of the alpha-amylase superfamily of enzymes, amylomaltase catalyzes either the transglycosylation from one alpha-1,4 glucan to another or an intramolecular cyclization. The latter reaction is typical for cyclodextrin glucanotransferases. In contrast to these enzymes, amylomaltase catalyzes the formation of cyclic glucans with a degree of polymerization larger than 22. To characterize the factors that determine the size of the synthesized cycloamyloses, we have analyzed the X-ray structure of amylomaltase from Thermus aquaticus in complex with the inhibitor acarbose, a maltotetraose derivative, at 1.9 A resolution. Two acarbose molecules are bound to the enzyme, one in the active site groove at subsite -3 to +1 and a second one approximately 14 A away from the nonreducing end of the acarbose bound to the catalytic site. The inhibitor bound to the catalytic site occupies subsites -3 to +1. Unlike the situation in other enzymes of the alpha-amylase family, the inhibitor is not processed and the inhibitory cyclitol ring of acarbose, which mimicks the half chair conformation of the transition state, does not bind to catalytic subsite -1. The minimum ring size of cycloamyloses produced by this enzyme is proposed to be determined by the distance of the specific substrate binding sites at the active site and near Tyr54 and by the size of the 460s loop. The 250s loop might be involved in binding of the substrate at the reducing end of the scissile bond.