The structure of MadC from Clostridium maddingley reveals new insights into class I lanthipeptide cyclases.

The rapid emergence of microbial multi-resistance against antibiotics has led to intense search for alternatives. One of these alternatives are ribosomally synthesized and post-translationally modified peptides (RiPPs), especially lantibiotics. They are active in a low nanomolar range and their high stability is due to the presence of characteristic (methyl-) lanthionine rings, which makes them promising candidates as bacteriocides. However, innate resistance against lantibiotics exists in nature, emphasizing the need for artificial or tailor-made lantibiotics. Obviously, such an approach requires an in-depth mechanistic understanding of the modification enzymes, which catalyze the formation of (methyl-)lanthionine rings. Here, we determined the structure of a class I cyclase (MadC), involved in the modification of maddinglicin (MadA) via X-ray crystallography at a resolution of 1.7 Å, revealing new insights about the structural composition of the catalytical site. These structural features and substrate binding were analyzed by mutational analyses of the leader peptide as well as of the cyclase, shedding light into the mode of action of MadC.

A promiscuous ancestral enzyme´s structure unveils protein variable regions of the highly diverse metallo-ß-lactamase family.

The metallo-ß-lactamase fold is an ancient protein structure present in numerous enzyme families responsible for diverse biological processes. The crystal structure of the hyperthermostable crenarchaeal enzyme Igni18 from Ignicoccus hospitalis was solved at 2.3 Å and could resemble a possible first archetype of a multifunctional metallo-ß-lactamase. Ancestral enzymes at the evolutionary origin are believed to be promiscuous all-rounders. Consistently, Igni18´s activity can be cofactor-dependently directed from ß-lactamase to lactonase, lipase, phosphodiesterase, phosphotriesterase or phospholipase. Its core-domain is highly conserved within metallo-ß-lactamases from Bacteria, Archaea and Eukarya and gives insights into evolution and function of enzymes from this superfamily. Structural alignments with diverse metallo-ß-lactamase-fold-containing enzymes allowed the identification of Protein Variable Regions accounting for modulation of activity, specificity and oligomerization patterns. Docking of different substrates within the active sites revealed the basis for the crucial cofactor dependency of this enzyme superfamily.

The architecture of the diaminobutyrate acetyltransferase active site provides mechanistic insight into the biosynthesis of the chemical chaperone ectoine.

Ectoine is a solute compatible with the physiologies of both prokaryotic and eukaryotic cells and is widely synthesized by bacteria as an osmotic stress protectant. Because it preserves functional attributes of proteins and macromolecular complexes, it is considered a chemical chaperone and has found numerous practical applications. However, the mechanism of its biosynthesis is incompletely understood. The second step in ectoine biosynthesis is catalyzed by l-2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), which transfers the acetyl group from acetyl-CoA to EctB-formed l-2,4-diaminobutyrate (DAB), yielding N-γ-acetyl-l-2,4-diaminobutyrate (N-γ-ADABA), the substrate of ectoine synthase (EctC). Here, we report the biochemical and structural characterization of the EctA enzyme from the thermotolerant bacterium Paenibacillus lautus (Pl). We found that (Pl)EctA forms a homodimer whose enzyme activity is highly regiospecific by producing N-γ-ADABA but not the ectoine catabolic intermediate N-α-acetyl-l-2,4-diaminobutyric acid. High-resolution crystal structures of (Pl)EctA (at 1.2-2.2 Å resolution) (i) for its apo-form, (ii) in complex with CoA, (iii) in complex with DAB, (iv) in complex with both CoA and DAB, and (v) in the presence of the product N-γ-ADABA were obtained. To pinpoint residues involved in DAB binding, we probed the structure-function relationship of (Pl)EctA by site-directed mutagenesis. Phylogenomics shows that EctA-type proteins from both Bacteria and Archaea are evolutionarily highly conserved, including catalytically important residues. Collectively, our biochemical and structural findings yielded detailed insights into the catalytic core of the EctA enzyme that laid the foundation for unraveling its reaction mechanism.

Igni18, a novel metallo-hydrolase from the hyperthermophilic archaeon Ignicoccus hospitalis KIN4/I: cloning, expression, purification and X-ray analysis.

The hyperthermophilic crenarchaeon Ignicoccus hospitalis KIN4/I possesses at least 35 putative genes encoding enzymes that belong to the α/ß-hydrolase superfamily. One of those genes, the metallo-hydrolase-encoding igni18, was cloned and heterologously expressed in Pichia pastoris. The enzyme produced was purified in its catalytically active form. The recombinant enzyme was successfully crystallized and the crystal diffracted to a resolution of 2.3 Å. The crystal belonged to space group R32, with unit-cell parameters a = b = 67.42, c = 253.77 Å, α = ß = 90.0, γ = 120.0°. It is suggested that it contains one monomer of Igni18 within the asymmetric unit.

Biosynthesis of the Stress-Protectant and Chemical Chaperon Ectoine: Biochemistry of the Transaminase EctB.

Bacteria frequently adapt to high osmolarity surroundings through the accumulation of compatible solutes. Ectoine is a prominent member of these types of stress protectants and is produced via an evolutionarily conserved biosynthetic pathway beginning with the L-2,4-diaminobutyrate (DAB) transaminase (TA) EctB. Here, we studied EctB from the thermo-tolerant Gram-positive bacterium Paenibacillus lautus (Pl) and show that this tetrameric enzyme is highly tolerant to salt, pH, and temperature. During ectoine biosynthesis, EctB converts L-glutamate and L-aspartate-beta-semialdehyde into 2-oxoglutarate and DAB, but it also catalyzes the reverse reaction. Our analysis unravels that EctB enzymes are mechanistically identical to the PLP-dependent gamma-aminobutyrate TAs (GABA-TAs) and only differ with respect to substrate binding. Inspection of the genomic context of the ectB gene in P. lautus identifies an unusual arrangement of juxtapositioned genes for ectoine biosynthesis and import via an Ehu-type binding-protein-dependent ABC transporter. This operon-like structure suggests the operation of a highly coordinated system for ectoine synthesis and import to maintain physiologically adequate cellular ectoine pools under osmotic stress conditions in a resource-efficient manner. Taken together, our study provides an in-depth mechanistic and physiological description of EctB, the first enzyme of the ectoine biosynthetic pathway.

α-Aminoxy Oligopeptides: Synthesis, Secondary Structure, and Cytotoxicity of a New Class of Anticancer Foldamers.

α-Aminoxy peptides are peptidomimetic foldamers with high proteolytic and conformational stability. To gain an improved synthetic access to α-aminoxy oligopeptides we used a straightforward combination of solution- and solid-phase-supported methods and obtained oligomers that showed a remarkable anticancer activity against a panel of cancer cell lines. We solved the first X-ray crystal structure of an α-aminoxy peptide with multiple turns around the helical axis. The crystal structure revealed a right-handed 28 -helical conformation with precisely two residues per turn and a helical pitch of 5.8 Å. By 2D ROESY experiments, molecular dynamics simulations, and CD spectroscopy we were able to identify the 28 -helix as the predominant conformation in organic solvents. In aqueous solution, the α-aminoxy peptides exist in the 28 -helical conformation at acidic pH, but exhibit remarkable changes in the secondary structure with increasing pH. The most cytotoxic α-aminoxy peptides have an increased propensity to take up a 28 -helical conformation in the presence of a model membrane. This indicates a correlation between the 28 -helical conformation and the membranolytic activity observed in mode of action studies, thereby providing novel insights in the folding properties and the biological activity of α-aminoxy peptides.

Structure of the Response Regulator NsrR from Streptococcus agalactiae, Which Is Involved in Lantibiotic Resistance.

Lantibiotics are antimicrobial peptides produced by Gram-positive bacteria. Interestingly, several clinically relevant and human pathogenic strains are inherently resistant towards lantibiotics. The expression of the genes responsible for lantibiotic resistance is regulated by a specific two-component system consisting of a histidine kinase and a response regulator. Here, we focused on a response regulator involved in lantibiotic resistance, NsrR from Streptococcus agalactiae, and determined the crystal structures of its N-terminal receiver domain and C-terminal DNA-binding effector domain. The C-terminal domain exhibits a fold that classifies NsrR as a member of the OmpR/PhoB subfamily of regulators. Amino acids involved in phosphorylation, dimerization, and DNA-binding were identified and demonstrated to be conserved in lantibiotic resistance regulators. Finally, a model of the full-length NsrR in the active and inactive state provides insights into protein dimerization and DNA-binding.

Structural basis of lantibiotic recognition by the nisin resistance protein from Streptococcus agalactiae.

Lantibiotics are potent antimicrobial peptides. Nisin is the most prominent member and contains five crucial lanthionine rings. Some clinically relevant bacteria express membrane-associated resistance proteins that proteolytically inactivate nisin. However, substrate recognition and specificity of these proteins is unknown. Here, we report the first three-dimensional structure of a nisin resistance protein from Streptococcus agalactiae (SaNSR) at 2.2 Å resolution. It contains an N-terminal helical bundle, and protease cap and core domains. The latter harbors the highly conserved TASSAEM region, which lies in a hydrophobic tunnel formed by all domains. By integrative modeling, mutagenesis studies, and genetic engineering of nisin variants, a model of the SaNSR/nisin complex is generated, revealing that SaNSR recognizes the last C-terminally located lanthionine ring of nisin. This determines the substrate specificity of SaNSR and ensures the exact coordination of the nisin cleavage site at the TASSAEM region.

Overexpression, purification and crystallization of the response regulator NsrR involved in nisin resistance.

A number of Gram-positive bacteria produce a class of bacteriocins called `lantibiotics'. These lantibiotics are ribosomally synthesized peptides that possess high antimicrobial activity against Gram-positive bacteria, including clinically challenging pathogens, and are therefore potential alternatives to antibiotics. All lantibiotic producer strains and some Gram-positive nonproducer strains express protein systems to circumvent a suicidal effect or to become resistant, respectively. Two-component systems consisting of a response regulator and a histidine kinase upregulate the expression of these proteins. One of the best-characterized lantibiotics is nisin, which is produced by Lactococcus lactis and possesses bactericidal activity against various Gram-positive bacteria, including some human pathogenic strains. Within many human pathogenic bacterial strains inherently resistant to nisin, a response regulator, NsrR, has been identified which regulates the expression of proteins involved in nisin resistance. In the present study, an expression and purification protocol was established for the NsrR protein from Streptococcus agalactiae COH1. The protein was successfully crystallized using the vapour-diffusion method, resulting in crystals that diffracted X-rays to 1.4 Šresolution.

Overproduction, crystallization and X-ray diffraction data analysis of ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis.

Ectoine biosynthetic genes (ectABC) are widely distributed in bacteria. Microorganisms that carry them make copious amounts of ectoine as a cell protectant in response to high-osmolarity challenges. Ectoine synthase (EctC; EC 4.2.1.108) is the key enzyme for the production of this compatible solute and mediates the last step of ectoine biosynthesis. It catalyzes the ring closure of the cyclic ectoine molecule. A codon-optimized version of ectC from Sphingopyxis alaskensis (Sa) was used for overproduction of SaEctC protein carrying a Strep-tag II peptide at its carboxy-terminus. The recombinant SaEctC-Strep-tag II protein was purified to near-homogeneity from Escherichia coli cell extracts by affinity chromatography. Size-exclusion chromatography revealed that it is a dimer in solution. The SaEctC-Strep-tag II protein was crystallized using the sitting-drop vapour-diffusion method and crystals that diffracted to 1.0 Šresolution were obtained.

Overexpression, purification, crystallization and preliminary X-ray diffraction of the nisin resistance protein from Streptococcus agalactiae.

Nisin is a 34-amino-acid antimicrobial peptide produced by Lactococcus lactis belonging to the class of lantibiotics. Nisin displays a high bactericidal activity against various Gram-positive bacteria, including some human-pathogenic strains. However, there are some nisin-non-producing strains that are naturally resistant owing to the presence of the nsr gene within their genome. The encoded protein, NSR, cleaves off the last six amino acids of nisin, thereby reducing its bactericidal efficacy. An expression and purification protocol has been established for the NSR protein from Streptococcus agalactiae COH1. The protein was successfully crystallized using the vapour-diffusion method in hanging and sitting drops, resulting in crystals that diffracted X-rays to 2.8 and 2.2 Å, respectively.

Overexpression, crystallization and preliminary X-ray crystallographic analysis of the ectoine hydroxylase from Sphingopyxis alaskensis.

The ectoine hydroxylase (EctD) is a member of the non-haem-containing iron(II)- and 2-oxoglutarate-dependent dioxygenase superfamily. Its mononuclear iron centre is a prerequisite for the activity of this enzyme and promotes the O2-dependent oxidative decarboxylation of 2-oxoglutarate, which is coupled to a two-electron oxidation of the substrate ectoine to yield 5-hydroxyectoine. An expression and purification protocol for the EctD enzyme from Sphingopyxis alaskensis was developed and the protein was crystallized using the sitting-drop vapour-diffusion method. This resulted in two different crystal forms, representing the apo and iron-bound forms of the enzyme.

Purification, crystallization and preliminary X-ray crystallographic analysis of the transport unit of the monomeric autotransporter AIDA-I from Escherichia coli.

The adhesin involved in diffuse adherence (AIDA-I) from Escherichia coli belongs to the group of autotransporters, specifically the type Va secretion system (T5aSS). All autotransporter systems contain a C-terminal ß-domain, which forms a barrel-like structure in the outer membrane with a hydrophilic pore allowing passenger translocation across the outer membrane. The passenger domain harbours the biological activity in the extracellular space and functions, for example, as an adhesin, an enzyme and a toxin. The exact transport mechanism of passenger translocation across the outer membrane is not clear at present. Thus, structure determination of the transport unit of AIDA-I could provide new insights into the transport mechanism. Here, the purification, crystallization and preliminary X-ray crystallographic studies of the transport unit of AIDA-I are reported.

Structure of the corrinoid:coenzyme M methyltransferase MtaA from Methanosarcina mazei.

The zinc-containing corrinoid:coenzyme M methyltransferase MtaA is part of the methanol-coenzyme M-methyltransferase complex of Methanosarcina mazei. The whole complex consists of three subunits: MtaA, MtaB and MtaC. The MtaB-MtaC complex catalyses the cleavage of methanol (bound to MtaB) and the transfer of the methyl group onto the cobalt of cob(I)alamin (bound to MtaC). The MtaA-MtaC complex catalyses methyl transfer from methyl-cob(III)alamin (bound to MtaC) to coenzyme M (bound to MtaA). The crystal structure of the MtaB-MtaC complex from M. barkeri has previously been determined. Here, the crystal structures of MtaA from M. mazei in a substrate-free but Zn(2+)-bound state and in complex with Zn(2+) and coenzyme M (HS-CoM) are reported at resolutions of 1.8 and 2.1 Å, respectively. A search for homologous proteins revealed that MtaA exhibits 23% sequence identity to human uroporphyrinogen III decarboxylase, which has also the highest structural similarity (r.m.s.d. of 2.03 Å for 306 aligned amino acids). The main structural feature of MtaA is a TIM-barrel-like fold, which is also found in all other zinc enzymes that catalyse thiol-group alkylation. The active site of MtaA is situated at the narrow bottom of a funnel such that the thiolate group of HS-CoM points towards the Zn(2+) ion. The Zn(2+) ion in the active site of MtaA is coordinated tetrahedrally via His240, Cys242 and Cys319. In the substrate-free form the fourth ligand is Glu263. Binding of HS-CoM leads to exchange of the O-ligand of Glu263 for the S-ligand of HS-CoM with inversion of the zinc geometry. The interface between MtaA and MtaC for transfer of the methyl group from MtaC-bound methylcobalamin is most likely to be formed by the core complex of MtaB-MtaC and the N-terminal segment (a long loop containing three α-helices and a ß-hairpin) of MtaA, which is not part of the TIM-barrel core structure of MtaA.