Structural Basis for Nucleotide Binding and Reaction Catalysis in Mevalonate Diphosphate Decarboxylase

Mevalonate diphosphate decarboxylase (MDD) catalyzes the final step of the mevalonate pathway, the Mg²⁺-ATP dependent decarboxylation of mevalonate 5-diphosphate (MVAPP), producing isopentenyl diphosphate (IPP). Synthesis of IPP, an isoprenoid precursor molecule that is a critical intermediate in peptidoglycan and polyisoprenoid biosynthesis, is essential in Gram-positive bacteria (e.g., <i>Staphylococcus</i>, <i>Streptococcus</i>, and <i>Enterococcus</i> spp.), and thus the enzymes of the mevalonate pathway are ideal antimicrobial targets. MDD belongs to the GHMP superfamily of metabolite kinases that have been extensively studied for the past 50 years, yet the crystallization of GHMP kinase ternary complexes has proven to be difficult. To further our understanding of the catalytic mechanism of GHMP kinases with the purpose of developing broad spectrum antimicrobial agents that target the substrate and nucleotide binding sites, we report the crystal structures of wild-type and mutant (S192A and D283A) ternary complexes of <i>Staphylococcus epidermidis</i> MDD. Comparison of apo, MVAPP-bound, and ternary complex wild-type MDD provides structural information about the mode of substrate binding and the catalytic mechanism. Structural characterization of ternary complexes of catalytically deficient MDD S192A and D283A (<i>k</i>_cat decreased 10³- and 10⁵-fold, respectively) provides insight into MDD function. The carboxylate side chain of invariant Asp²⁸³ functions as a catalytic base and is essential for the proper orientation of the MVAPP C3-hydroxyl group within the active site funnel. Several MDD amino acids within the conserved phosphate binding loop (“P-loop”) provide key interactions, stabilizing the nucleotide triphosphoryl moiety. The crystal structures presented here provide a useful foundation for structure-based drug design

Enantioselective Synthesis of Dilignol Model Compounds and Their Stereodiscrimination Study with a Dye-Decolorizing Peroxidase

A four-step enantioselective approach was developed to synthesize <i>anti</i> (1<i>R</i>,2<i>S</i>)-<b>1a</b> and (1<i>S</i>,2<i>R</i>)-<b>1b</b> containing a β-O-4 linkage in good yields. A significant difference was observed for the apparent binding affinities of four stereospecific lignin model compounds with <i>Tc</i>DyP by surface plasmon resonance, which was not translated into a significant difference in enzyme activities. The discrepancy may be attributed to the conformational change involving a loop widely present in DyPs upon H₂O₂ binding

Mechanistic Insights into Dye-Decolorizing Peroxidase Revealed by Solvent Isotope and Viscosity Effects

Dye-decolorizing peroxidases (DyPs) are a family of H₂O₂-dependent heme peroxidases that have shown potential applications in lignin degradation and valorization. However, the DyP kinetic mechanism remains underexplored. Using structural biology and solvent isotope (sKIE) and viscosity effects, many mechanistic characteristics have been determined for the B-class <i>El</i>DyP from <i>Enterobacter lignolyticus</i>. Its structure revealed that a water molecule acts as the sixth axial ligand and two channels at diameters of ∼3.0 and 8.0 Å lead to the heme center. A conformational change of ERS* to ERS, which have identical spectral characteristics, was proposed as the final step in DyPs’ bisubstrate Ping-Pong mechanism. This step is also the rate-determining step in ABTS oxidation. The normal KIE of wild-type <i>El</i>DyP with D₂O₂ at pD 3.5 suggested that compound 0 deprotonation by the distal aspartate is rate-limiting in the formation of compound I, which is more reactive under acidic pH than under neutral or alkaline pH. The viscosity effects and other biochemical methods implied that the reducing substrate binds with compound I instead of the free enzyme. The significant inverse sKIEs of <i>k</i>_cat/<i>K</i>_M and <i>k</i>_ERS* suggested that the aquo release in <i>El</i>DyP is mechanistically important and may explain the enzyme’s adoption of two-electron reduction for compound I. The distal aspartate is catalytically more important than the distal arginine and plays key roles in determining <i>El</i>DyP’s optimum acidic pH. The kinetic mechanism of D143H-<i>El</i>DyP was also briefly studied. The results obtained will pave the way for future protein engineering to improve DyPs’ lignolytic activity

Binding Affects the Tertiary and Quaternary Structures of the <i>Shigella</i> Translocator Protein IpaB and Its Chaperone IpgC

<i>Shigella flexneri</i> uses its type III secretion system (T3SS) to promote invasion of human intestinal epithelial cells as the first step in causing shigellosis, a life-threatening form of dysentery. The <i>Shigella</i> type III secretion apparatus (T3SA) consists of a basal body that spans the bacterial envelope and an exposed needle that injects effector proteins into target cells. The nascent <i>Shigella</i> T3SA needle is topped with a pentamer of the needle tip protein invasion plasmid antigen D (IpaD). Bile salts trigger recruitment of the first hydrophobic translocator protein, IpaB, to the tip complex where it senses contact with a host membrane. In the bacterial cytoplasm, IpaB exists in a complex with its chaperone IpgC. Several structures of IpgC have been determined, and we recently reported the 2.1 Å crystal structure of the N-terminal domain (IpaB^74.224) of IpaB. Like IpgC, the IpaB N-terminal domain exists as a homodimer in solution. We now report that when the two are mixed, these homodimers dissociate and form heterodimers having a nanomolar dissociation constant. This is consistent with the equivalent complexes copurified after they had been co-expressed in <i>Escherichia coli</i>. Fluorescence data presented here also indicate that the N-terminal domain of IpaB possesses two regions that appear to contribute additively to chaperone binding. It is also likely that the N-terminus of IpaB adopts an alternative conformation as a result of chaperone binding. The importance of these findings within the functional context of these proteins is discussed

Identification of Surface-Exposed Protein Radicals and A Substrate Oxidation Site in A‑Class Dye-Decolorizing Peroxidase from <i>Thermomonospora curvata</i>

Dye-decolorizing peroxidases (DyPs) are a family of heme peroxidases in which a catalytic distal aspartate is involved in H₂O₂ activation to catalyze oxidations under acidic conditions. They have received much attention due to their potential applications in lignin compound degradation and biofuel production from biomass. However, the mode of oxidation in bacterial DyPs remains unknown. We have recently reported that the bacterial <i>Tc</i>DyP from Thermomonospora curvata is among the most active DyPs and shows activity toward phenolic lignin model compounds. On the basis of the X-ray crystal structure solved at 1.75 Å, sigmoidal steady-state kinetics with Reactive Blue 19 (RB19), and formation of compound II like product in the absence of reducing substrates observed with stopped-flow spectroscopy and electron paramagnetic resonance (EPR), we hypothesized that the <i>Tc</i>DyP catalyzes oxidation of large-size substrates via multiple surface-exposed protein radicals. Among 7 tryptophans and 3 tyrosines in <i>Tc</i>DyP consisting of 376 residues for the matured protein, W263, W376, and Y332 were identified as surface-exposed protein radicals. Only the W263 was also characterized as one of the surface-exposed oxidation sites. SDS-PAGE and size-exclusion chromatography demonstrated that W376 represents an off-pathway destination for electron transfer, resulting in the cross-linking of proteins in the absence of substrates. Mutation of W376 improved compound I stability and overall catalytic efficiency toward RB19. While Y332 is highly conserved across all four classes of DyPs, its catalytic function in A-class <i>Tc</i>DyP is minimal, possibly due to its extremely small solvent-accessible areas. Identification of surface-exposed protein radicals and substrate oxidation sites is important for understanding the DyP mechanism and modulating its catalytic functions for improved activity on phenolic lignin

Image_1_The Staphylococcus aureus Extracellular Adherence Protein Eap Is a DNA Binding Protein Capable of Blocking Neutrophil Extracellular Trap Formation.PDF

<p>The extracellular adherence protein (Eap) of Staphylococcus aureus is a secreted protein known to exert a number of adhesive and immunomodulatory properties. Here we describe the intrinsic DNA binding activity of this multifunctional secretory factor. By using atomic force microscopy, we provide evidence that Eap can bind and aggregate DNA. While the origin of the DNA substrate (e.g., eukaryotic, bacterial, phage, and artificial DNA) seems to not be of major importance, the DNA structure (e.g., linear or circular) plays a critical role with respect to the ability of Eap to bind and condense DNA. Further functional assays corroborated the nature of Eap as a DNA binding protein, since Eap suppressed the formation of “neutrophil extracellular traps” (NETs), composed of DNA-histone scaffolds, which are thought to function as a neutrophil-mediated extracellular trapping mechanism. The DNA binding and aggregation activity of Eap may thereby protect S. aureus against a specific anti-microbial defense reaction from the host.</p