SUMMARY 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 α-dimethylbutyryl thioester, albeit with suboptimal properties as a biocatalyst. Here we used directed evolution to improve the properties of LovD towards semisynthesis of simvastatin. Mutants with improved catalytic efficiency, solubility and thermal stability were obtained, with the best mutant displaying an ~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 co-crystallized 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.
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 (MARTXVc), which contributes significantly to the pathogenesis of cholera in model systems. The actin cross-linking domain (ACD) of MARTXVc 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.cross-linking ͉ X-ray crystallography ͉ mass spectrometry A s a vital component of every eukaryotic cell, the actin cytoskeleton is a frequent target for microbial toxins. By impairing actin-orchestrated functions via production of specific toxins, pathogenic bacteria manage to escape immune cell surveillance and not only overcome cell barriers but also hijack the actin cytoskeleton for efficient invasion and dissemination. These toxins modulate activities of host proteins either via direct binding to or covalent modifications of their targets. Most of the toxins acting on the actin cytoskeleton shift the equilibrium between polymerized F-and monomeric G-actin by targeting actin adaptor and regulatory proteins such as the Arp2/3 complex and RhoGTPases (1). A lower number of protein toxins affect actin directly rather than through other components of the cytoskeleton. Two types of covalent modifications of actin by two distinct groups of bacterial toxins have been described so far. The first group consists of a family of interrelated toxins with ADP-ribosyltransferase activity (such as C2 toxin of Clostridium botulinum, Iota toxin of C. perfringens, and SpvB of Salmonella enterica), which block actin polymerization by adding an ADPribose moiety to Arg-177 of G-actin, thereby resulting in sterical clashes between actin protomers (2, 3).The second group is represented by MARTX Vc [multifunctional-autoprocessing repeats-in-toxins (RTX) toxin of V. cholerae], a toxin produced by nearly all environment...
SUMMARY 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 green fluorescent protein (GFP) as a scaffold, and engineered many alternate 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.
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.
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