X-ray crystallographic analysis of homooligomeric flavin containing cysteine decarboxylases

Die Familie der homooligomeren Flavin-enthaltenden Cystein-Decarboxylasen (HFCD) kann in zwei Unterfamilien eingeteilt werden. Die in dieser Arbeit untersuchten Proteine EpiD und MrsD gehören zusammen mit MutD zu den LanD-Proteinen. Diese katalysieren einen wichtigen Reaktionsschritt, eine oxidative Decarboxylierung eines C-terminalen Cysteins, in der Biosynthese der Lantibiotika Epidermin, Mersacidin und Mutacin III. Zwei weitere Mitglieder der HFCD-Proteine, das bakterielle Dfp und HAL3 aus Arabidopsis thaliana (AtHAL3) katalysieren die nicht oxidative Decarboxylierung von (R)-4'-Phospho-N-Panthenoylcystein zu 4'-Phosphopantethein in der Biosynthese des bei allen Organismen sehr wichtigen Coenzyms A (CoA). Die Kristallstrukturen der Proteine EpiD und MrsD sowie der inaktiven Mutante EpiD-H67N im Komplex mit dem Pentapeptid DSYTC wurden gelöst. Die Homododekamere der untersuchten Proteine bilden Partikel mit einer 23-Punktsymmetrie, mit auf den Spitzen eines Tetraeders lokalisierten Trimeren. Die Monomere besitzen eine Rossmann-artige Tertiärstruktur, wobei sich die Wechselwirkungen dieser Tertiärstruktur, die häufig von dinukleotidbindenden Proteinen verwendet wird, mit den Kofaktoren FMN und FAD bei den HFCD-Proteinen deutlich von denen anderer Flavoproteine unterscheiden. Die beiden charakteristischen Sequenzmotive dieser Proteinfamilie, PASANT und PXMNXXMW, sind an der Kofaktorbindung, Wechselwirkungen mit dem Substrat und wichtigen strukturellen Eigenschaften beteiligt. Die Struktur des Komplexes von EpiD-H67N mit DSYTC und ein modellierter Komplex von MrsD mit den fünf C-terminalen Aminosäuren des Vorläuferpeptides von Mersacidin (MrsA) erklären die unüblich breite Substratspezifität von EpiD und die Unterschiede zu derjenigen von MrsD. Die von den Proteinen der HFCD-Familie katalysierte Decarboxylierung eines C-terminalen Cysteins zeigt einen neuartigen Reaktionsmechanismus, der sich deutlich von den bisher bekannten Decarboxylierungen und Dehydrierungen unterscheidet. Als ersten Reaktionsschritt kann man eine Oxidation der Thiolgruppe annehmen, da nur diese Kontakt mit dem Kofaktor hat. Die entstehende beta-Thioaldehyd-Carbonsäure decarboxyliert danach analog zu den beta-Keto-Carbonsäuren wahrscheinlich spontan.The family of homooligomeric flavin containing cystein decarboxylases (HFCD) can be divided into two subfamilies. The proteins investigated in this work, EpiD and MrsD belong together with MutD to the so-called LanD proteins. They catalyse an important reaction step, an oxidative decarboxylation of a C-terminal cysteine, during the biosynthesis of the lantibiotics epidermin, mersacidin and mutacin III, respectively. Two further members of the HFCD proteins, the bacterial Dfp and HAL3 from Arabidopsis thaliana (AtHAL3) catalyse the non-oxidative decarboxylation of (R)-4'-phospho-N-panthenoylcysteine to 4'-Phosphopantetheine during the biosynthesis of coenzyme A, which is very important for all organisms. The crystal structure of the proteins EpiD and MrsD, as well as the inactive mutant EpiD-H67N complexed with the pentapeptide DSYTC could be solved. The homododecamers of the investigated proteins build up particles of 23 point symmetry, with trimers localised at the vertices of a tetrahedron. The protomers show a typical Rossmann fold but the interactions of this tertiary structure, which is used often by dinucleotide binding proteins, with the cofactors FMN and FAD within the HFCD family differs clearly from that of other flavoproteins. The two characteristic sequence motifs PASANT and PXMNXXWM of this family are involved in binding of the cofactor and substrate as well as important structural features. The structure of the complex of EpiD with DSYTC and a modeled complex of MrsD with the five C-terminal amino acids of the precursor of mersacidin MrsA explain the unusually broad substrate specificity of EpiD and the differences to MrsD. The catalysed decarboxylation of a C-terminal cysteine shows a novel mechanism, which differs clearly from the known decarboxylations and dehydrogenations. As a first step one could suggest the oxidation of the thiol group, because it is the only one which is in contact with the cofactor. The evolving beta-thioaldehyd might decarboxylate spontaneously like the analogous beta-ketocarbonic acids

Carbonic anhydrase seven bundles filamentous actin and regulates dendritic spine morphology and density

Intracellular pH is a potent modulator of neuronal functions. By catalyzing (de)hydration of CO2, intracellular carbonic anhydrase (CA(i)) isoforms CA2 and CA7 contribute to neuronal pH buffering and dynamics. The presence of two highly active isoforms in neurons suggests that they may serve isozyme-specific functions unrelated to CO2-(de)hydration. Here, we show that CA7, unlike CA2, binds to filamentous actin, and its overexpression induces formation of thick actin bundles and membrane protrusions in fibroblasts. In CA7-overexpressing neurons, CA7 is enriched in dendritic spines, which leads to aberrant spine morphology. We identified amino acids unique to CA7 that are required for direct actin interactions, promoting actin filament bundling and spine targeting. Disruption of CA7 expression in neocortical neurons leads to higher spine density due to increased proportion of small spines. Thus, our work demonstrates highly distinct subcellular expression patterns of CA7 and CA2, and a novel, structural role of CA7.Peer reviewe

Crystal structure of ATP sulfurylase from Saccharomyces cerevisiae, a key enzyme in sulfate activation

ATP sulfurylases (ATPSs) are ubiquitous enzymes that catalyse the primary step of intracellular sulfate activation: the reaction of inorganic sulfate with ATP to form adenosine-5′-phosphosulfate (APS) and pyrophosphate (PPi). With the crystal structure of ATPS from the yeast Saccharomyces cerevisiae, we have solved the first structure of a member of the ATP sulfurylase family. We have analysed the crystal structure of the native enzyme at 1.95 Å resolution using multiple isomorphous replacement (MIR) and, subsequently, the ternary enzyme product complex with APS and PPi bound to the active site. The enzyme consists of six identical subunits arranged in two stacked rings in a D3 symmetric assembly. Nucleotide binding causes significant conformational changes, which lead to a rigid body structural displacement of domains III and IV of the ATPS monomer. Despite having similar folds and active site design, examination of the active site of ATPS and comparison with known structures of related nucleotidylyl transferases reveal a novel ATP binding mode that is peculiar to ATP sulfuryl-ases

Structure of MrsD, an FAD-binding protein of the HFCD family

MrsD from Bacillus sp. HIL-Y85/54728 is a member of the HFCD (homo-oligomeric flavin-containing Cys decarboxylases) family of flavoproteins and is involved in the biosynthesis of the lantibiotic mersacidin. It catalyses the oxidative decarboxylation of the C-terminal cysteine residue of the MrsA precursor peptide of mersacidin, yielding a (Z)-enethiol intermediate as the first step in the formation of the unusual amino acid S-[(Z)-2-aminovinyl]-methyl-D-cysteine. Surprisingly, MrsD was found to bind FAD, in contrast to the three other characterized members of the HFCD family, which bind FMN. To determine the molecular discriminators of FAD binding within the HFCD family, the crystal structure of MrsD was analyzed at a resolution of 2.54 Å. Crystals of space group F432 contain one MrsD monomer in the asymmetric unit. However, a Patterson search with EpiD-derived models failed. Based on the consideration that the dodecameric MrsD particle of tetrahedral symmetry resembles the quaternary structure of EpiD, rotational and translational parameters were derived from the geometric consideration that the MrsD dodecamer is generated from a monomer by crystallographic symmetry around the position (1/4, 1/4, 1/4) of the unit cell. A structural comparison with the FMN-binding members of the HFCD family EpiD and AtHAL3a shows conserved sequence motifs in contact with the flavin's pyrimidine ring but divergent environments for the dimethylbenzene ring of the isoalloxazine moiety. The position of the ribityl chain differs in MrsD from that found in EpiD and AtHAL3a. However, the FMN-phosphate binding sites are also highly conserved in their exact positions. In all three cases, the flavin cofactor is bound to a structurally conserved region of the Rossmann-fold monomer, exposing its Re side for catalysis. The adenosyl phosphate of FAD is anchored in a well defined binding site and the adenosine moieties are oriented towards the interior of the hollow particle, where three of them pack against each other around the threefold axis of a trimeric facet

Crystal structure of the peptidyl-cysteine decarboxylase EpiD complexed with a pentapeptide substrate

Epidermin from Staphylococcus epidermidis Tü3298 is an antimicrobial peptide of the lantibiotic family that contains, amongst other unusual amino acids, S-[(Z)- 2-aminovinyl]-d-cysteine. This residue is introduced by post-translational modification of the ribosomally synthesized precursor EpiA. Modification starts with the oxidative decarboxylation of its C-terminal cysteine by the flavoprotein EpiD generating a reactive (Z)-enethiol intermediate. We have determined the crystal structures of EpiD and EpiD H67N in complex with the substrate pentapeptide DSYTC at 2.5 Å resolution. Rossmann-type monomers build up a dodecamer of 23 point symmetry with trimers disposed at the vertices of a tetrahedron. Oligomer formation is essential for binding of flavin mononucleotide and substrate, which is buried by an otherwise disordered substrate recognition clamp. A pocket for the tyrosine residue of the substrate peptide is formed by an induced fit mechanism. The substrate contacts flavin mononucleotide only via Cys-Sγ, suggesting its oxidation as the initial step. A thioaldehyde intermediate could undergo spontaneous decarboxylation. The unusual substrate recognition mode and the type of chemical reaction performed provide insight into a novel family of flavoproteins

Molecular Insights into Function and Competitive Inhibition of Pseudomonas aeruginosa Multiple Virulence Factor Regulator

ABSTRACT New approaches to antimicrobial drug discovery are urgently needed to combat intractable infections caused by multidrug-resistant (MDR) bacteria. Multiple virulence factor regulator (MvfR or PqsR), a Pseudomonas aeruginosa quorum sensing transcription factor, regulates functions important in both acute and persistent infections. Recently identified non-ligand-based benzamine-benzimidazole (BB) inhibitors of MvfR suppress both acute and persistent P. aeruginosa infections in mice without perturbing bacterial growth. Here, we elucidate the crystal structure of the MvfR ligand binding domain (LBD) in complex with one potent BB inhibitor, M64. Structural analysis indicated that M64 binds, like native ligands, to the MvfR hydrophobic cavity. A hydrogen bond and pi interaction were found to be important for MvfR-M64 affinity. Surface plasmon resonance analysis demonstrated that M64 is a competitive inhibitor of MvfR. Moreover, a protein engineering approach revealed that Gln194 and Tyr258 are critical for the interaction between MvfR and M64. Random mutagenesis of the full-length MvfR protein identified a single-amino-acid substitution, I68F, at a DNA binding linker domain that confers M64 insensitivity. In the presence of M64, I68F but not the wild-type (WT) MvfR protein retained DNA binding ability. Our findings strongly suggest that M64 promotes conformational change at the DNA binding domain of MvfR and that the I68F mutation may compensate for this change, indicating allosteric inhibition. This work provides critical new insights into the molecular mechanism of MvfR function and inhibition that could aid in the optimization of anti-MvfR compounds and improve our understanding of MvfR regulation