Structural and biochemical characterization of the salicylyl-acyltranferase SsfX3 from a tetracycline biosynthetic pathway.

SsfX3 is a GDSL family acyltransferase that transfers salicylate to the C-4 hydroxyl of a tetracycline intermediate in the penultimate step during biosynthesis of the anticancer natural product SF2575. The C-4 salicylate takes the place of the more common C-4 dimethylamine functionality, making SsfX3 the first acyltransferase identified to act on a tetracycline substrate. The crystal structure of SsfX3 was determined at 2.5 Å, revealing two distinct domains as follows: an N-terminal ß-sandwich domain that resembles a carbohydrate-binding module, and a C-terminal catalytic domain that contains the atypical α/ß-hydrolase fold found in the GDSL hydrolase family of enzymes. The active site lies at one end of a large open binding pocket, which is spatially defined by structural elements from both the N- and C-terminal domains. Mutational analysis in the putative substrate binding pocket identified residues from both domains that are important for binding the acyl donor and acceptor. Furthermore, removal of the N-terminal carbohydrate-binding module-like domain rendered the stand-alone α/ß-hydrolase domain inactive. The additional noncatalytic module is therefore proposed to be required to define the binding pocket and provide sufficient interactions with the spatially extended tetracyclic substrate. SsfX3 was also demonstrated to accept a variety of non-native acyl groups. This relaxed substrate specificity toward the acyl donor allowed the chemoenzymatic biosynthesis of C-4-modified analogs of the immediate precursor to the bioactive SF2575; these were used to assay the structure activity relationships at the C-4 position.

Connecting actin monomers by iso-peptide bond is a toxicity mechanism of the Vibrio cholerae MARTX toxin.

The Gram-negative bacterium Vibrio cholerae is the causative agent of a severe diarrheal disease that afflicts three to five million persons annually, causing up to 200,000 deaths. Nearly all V. cholerae strains produce a large multifunctional-autoprocessing RTX toxin (MARTX(Vc)), which contributes significantly to the pathogenesis of cholera in model systems. The actin cross-linking domain (ACD) of MARTX(Vc) directly catalyzes a covalent cross-linking of monomeric G-actin into oligomeric chains and causes cell rounding, but the nature of the cross-linked bond and the mechanism of the actin cytoskeleton disruption remained elusive. To elucidate the mechanism of ACD action and effect on actin, we identified the covalent cross-link bond between actin protomers using limited proteolysis, X-ray crystallography, and mass spectrometry. We report here that ACD catalyzes the formation of an intermolecular iso-peptide bond between residues E270 and K50 located in the hydrophobic and the DNaseI-binding loops of actin, respectively. Mutagenesis studies confirm that no other residues on actin can be cross-linked by ACD both in vitro and in vivo. This cross-linking locks actin protomers into an orientation different from that of F-actin, resulting in strong inhibition of actin polymerization. This report describes a microbial toxin mechanism acting via iso-peptide bond cross-linking between host proteins and is, to the best of our knowledge, the only known example of a peptide linkage between nonterminal glutamate and lysine side chains.

Rational improvement of simvastatin synthase solubility in Escherichia coli leads to higher whole-cell biocatalytic activity.

Simvastatin is the active pharmaceutical ingredient of the blockbuster cholesterol lowering drug Zocor. We have previously developed an Escherichia coli based whole-cell biocatalytic platform towards the synthesis of simvastatin sodium salt (SS) starting from the precursor monacolin J sodium salt (MJSS). The centerpiece of the biocatalytic approach is the simvastatin synthase LovD, which is highly prone to misfolding and aggregation when overexpressed from E. coli. Increasing the solubility of LovD without decreasing its catalytic activity can therefore elevate the performance of the whole-cell biocatalyst. Using a combination of homology structural prediction and site-directed mutagenesis, we identified two cysteine residues in LovD that are responsible for nonspecific intermolecular crosslinking, which leads to oligomer formation and protein aggregation. Replacement of Cys40 and Cys60 with alanine residues resulted in marked gain in both protein solubility and whole-cell biocatalytic activities. Further mutagenesis experiments converting these two residues to small or polar natural amino acids showed that C40A and C60N are the most beneficial, affording 27% and 26% increase in whole cell activities, respectively. The double mutant C40A/C60N combines the individual improvements and displayed approximately 50% increase in protein solubility and whole-cell activity. Optimized fed-batch high-cell-density fermentation of the double mutant in an E. coli strain engineered for simvastatin production quantitatively (>99%) converted 45 mM MJSS to SS within 18 h, which represents a significant improvement over the performance of wild-type LovD under identical conditions. The high efficiency of the improved whole-cell platform renders the biocatalytic synthesis of SS an attractive substitute over the existing semisynthetic routes.

Crystal structure of the Mycobacterium tuberculosis dUTPase: insights into the catalytic mechanism.

The structure of Mycobacterium tuberculosis dUTP nucleotidohydrolase (dUTPase) has been determined at 1.3 Angstrom resolution in complex with magnesium ion and the non-hydrolyzable substrate analog, alpha,beta-imido dUTP. dUTPase is an enzyme essential for depleting potentially toxic concentrations of dUTP in the cell. Given the importance of its biological role, it has been proposed that inhibiting M.tuberculosis dUTPase might be an effective means to treat tuberculosis infection in humans. The crystal structure presented here offers some insight into the potential for designing a specific inhibitor of the M.tuberculosis dUTPase enzyme. The structure also offers new insights into the mechanism of dUTP hydrolysis by providing an accurate representation of the enzyme-substrate complex in which both the metal ion and dUTP analog are included. The structure suggests that inclusion of a magnesium ion is important for stabilizing the position of the alpha-phosphorus for an in-line nucleophilic attack. In the absence of magnesium, the alpha-phosphate of dUTP can have either of the two positions which differ by 4.5 Angstrom. A transiently ordered C-terminal loop further assists catalysis by shielding the general base, Asp83, from solvent thus elevating its pK(a) so that it might in turn activate a tightly bound water molecule for nucleophilic attack. The metal ion coordinates alpha, beta, and gamma phosphate groups with tridentate geometry identical with that observed in the crystal structure of DNA polymerase beta complexed with magnesium and dNTP analog, revealing some common features in catalytic mechanism.

A Suite of Engineered GFP Molecules for Oligomeric Scaffolding.

Applications ranging from synthetic biology to protein crystallization could be advanced by facile systems for connecting multiple proteins together in predefined spatial relationships. One approach to this goal is to engineer many distinct assembly forms of a single carrier protein or scaffold, to which other proteins of interest can then be readily attached. In this work we chose GFP as a scaffold and engineered many alternative oligomeric forms, driven by either specific disulfide bond formation or metal ion addition. We generated a wide range of spatial arrangements of GFP subunits from 11 different oligomeric variants, and determined their X-ray structures in a total of 33 distinct crystal forms. Some of the oligomeric GFP variants show geometric polymorphism depending on conditions, while others show considerable geometric rigidity. Potential future applications of this system are discussed.

Directed evolution and structural characterization of a simvastatin synthase.

Enzymes from natural product biosynthetic pathways are attractive candidates for creating tailored biocatalysts to produce semisynthetic pharmaceutical compounds. LovD is an acyltransferase that converts the inactive monacolin J acid (MJA) into the cholesterol-lowering lovastatin. LovD can also synthesize the blockbuster drug simvastatin using MJA and a synthetic alpha-dimethylbutyryl thioester, albeit with suboptimal properties as a biocatalyst. Here we used directed evolution to improve the properties of LovD toward semisynthesis of simvastatin. Mutants with improved catalytic efficiency, solubility, and thermal stability were obtained, with the best mutant displaying an approximately 11-fold increase in an Escherichia coli-based biocatalytic platform. To understand the structural basis of LovD enzymology, seven X-ray crystal structures were determined, including the parent LovD, an improved mutant G5, and G5 cocrystallized with ligands. Comparisons between the structures reveal that beneficial mutations stabilize the structure of G5 in a more compact conformation that is favorable for catalysis.

Multiple crystal structures of actin dimers and their implications for interactions in the actin filament.

The structure of actin in its monomeric form is known at high resolution, while the structure of filamentous F-actin is only understood at considerably lower resolution. Knowing precisely how the monomers of actin fit together would lead to a deeper understanding of the dynamic behavior of the actin filament. Here, a series of crystal structures of actin dimers are reported which were prepared by cross-linking in either the longitudinal or the lateral direction in the filament state. Laterally cross-linked dimers, comprised of monomers belonging to different protofilaments, are found to adopt configurations in crystals that are not related to the native structure of filamentous actin. In contrast, multiple structures of longitudinal dimers consistently reveal the same interface between monomers within a single protofilament. The reappearance of the same longitudinal interface in multiple crystal structures adds weight to arguments that the interface visualized is similar to that in actin filaments. Highly conserved atomic interactions involving residues 199-205 and 287-291 are highlighted.

The 1.70 angstroms X-ray crystal structure of Mycobacterium tuberculosis phosphoglycerate mutase.

The single-crystal X-ray structure of phosphoglycerate mutase from Mycobacterium tuberculosis has been determined at a resolution of 1.70 angstroms. The C-terminal tail of each of the subunits is flexible and disordered; however, for one of the four chains (chain A) all but five residues of the chain could be modeled. Noteworthy features of the structure include the active site and a proline-rich segment in each monomer forming a short left-handed polyprolyl helix. These segments lie on the enzyme surface and could conceivably participate in protein-protein interactions.