Claudia Roach

Lovastatin biosynthesis in Aspergillus terreus: Characterization of blocked mutants, enzyme activities and a multifunctional polyketide synthase gene

BACKGROUND Lovastatin, an HMG-CoA reductase inhibitor produced by the fungus Aspergillus terreus, is composed of two polyketide chains. One is a nonaketide that undergoes cyclization to a hexahydronaphthalene ring system and the other is a simple diketide, 2-methylbutyrate. Fungal polyketide synthase (PKS) systems are of great interest and their genetic manipulation should lead to novel compounds. RESULTS An A. terreus mutant (BX102) was isolated that could not synthesize the nonaketide portion of lovastatin and was missing a approximately 250 kDa polypeptide normally present under conditions of lovastatin production. Other mutants produced lovastatin intermediates without the methylbutyryl sidechain and were missing a polypeptide of approximately 220 kDa. The PKS inhibitor cerulenin reacted covalently with both polypeptides. Antiserum raised against the approximately 250 kDa polypeptide was used to isolate the corresponding gene, which complemented the BX102 mutation. The gene encodes a polypeptide of 269 kDa containing catalytic domains typical of vertebrate fatty acid and fungal PKSs, plus two additional domains not previously seen in PKSs: a centrally located methyltransferase domain and a peptide synthetase elongation domain at the carboxyl terminus. CONCLUSIONS The results show that the nonaketide and diketide portions of lovastatin are synthesized by separate large multifunctional PKSs. Elucidation of the primary structure of the PKS that forms the lovastatin nonaketide, as well as characterization of blocked mutants, provides new details of lovastatin biosynthesis.

Aldolase provides an unusual binding site for thrombospondin-related anonymous protein in the invasion machinery of the malaria parasite

An actomyosin motor located underneath the plasma membrane drives motility and host-cell invasion of apicomplexan parasites such as Plasmodium falciparum and Plasmodium vivax, the causative agents of malaria. Aldolase connects the motor actin filaments to transmembrane adhesive proteins of the thrombospondin-related anonymous protein (TRAP) family and transduces the motor force across the parasite surface. The TRAP–aldolase interaction is a distinctive and critical trait of host hepatocyte invasion by Plasmodium sporozoites, with a likely similar interaction crucial for erythrocyte invasion by merozoites. Here, we describe 2.4-Å and 2.7-Å structures of P. falciparum aldolase (PfAldo) obtained from crystals grown in the presence of the C-terminal hexapeptide of TRAP from Plasmodium berghei. The indole ring of the critical penultimate Trp-residue of TRAP fits snugly into a newly formed hydrophobic pocket, which is exclusively delimited by hydrophilic residues: two arginines, one glutamate, and one glutamine. Comparison with the unliganded PfAldo structure shows that the two arginines adopt new side-chain rotamers, whereas a 25-residue subdomain, forming a helix–loop–helix unit, shifts upon binding the TRAP-tail. The structural data are in agreement with decreased TRAP binding after mutagenesis of PfAldo residues in and near the induced TRAP-binding pocket. Remarkably, the TRAP- and actin-binding sites of PfAldo seem to overlap, suggesting that both the plasticity of the aldolase active-site region and the multimeric nature of the enzyme are crucial for its intriguing nonenzymatic function in the invasion machinery of the malaria parasite.

Crystal structures, metal activation, and DNA-binding properties of two-domain IdeR from Mycobacterium tuberculosis.

The iron-dependent regulator IdeR is a key transcriptional regulator of iron uptake in Mycobacterium tuberculosis. In order to increase our insight into the role of the SH3-like third domain of this essential regulator, the metal-binding and DNA-binding properties of two-domain IdeR (2D-IdeR) whose SH3-like domain has been truncated were characterized. The equilibrium dissociation constants for Co2+ and Ni2+ activation of 2D-IdeR for binding to the fxbA operator and the DNA-binding affinities of 2D-IdeR in the presence of excess metal ions were estimated using fluorescence spectroscopy. 2D-IdeR binds to fxbA operator DNA with similar affinity as full-length IdeR in the presence of excess metal ion. However, the Ni2+ concentrations required to activate 2D-IdeR for DNA binding appear to be smaller than that for full-length IdeR while the concentration of Co2+ required for activation remains the same. We have determined the crystal structures of Ni2+-activated 2D-IdeR at 1.96 A resolution and its double dimer complex with the mbtA-mbtB operator DNA in two crystal forms at 2.4 A and 2.6 A, the highest resolutions for DNA complexes for any structures of iron-dependent regulator family members so far. The 2D-IdeR-DNA complex structures confirm the specificity of Ser37 and Pro39 for thymine bases and suggest preferential contacts of Gln43 to cytosine bases of the DNA. In addition, our 2D-IdeR structures reveal a remarkable property of the TEV cleavage sequence remaining after removal of the C-terminal His6. This C-terminal tail promotes crystal contacts by forming a beta-sheet with the corresponding tail of neighboring subunits in two unrelated structures of 2D-IdeR, one with and one without DNA. The contact-promoting properties of this C-terminal TEV cleavage sequence may be beneficial for crystallizing other proteins.

Exploration of the GM1 receptor-binding site of heat-labile enterotoxin and cholera toxin by phenyl-ring-containing galactose derivatives

Cholera toxin (CT) and the closely related heat-labile enterotoxin of Escherichia coli (LT) are responsible for numerous cases of diarrhea worldwide, leading to considerable morbidity and mortality. The B subunits of these heterohexameric AB(5) toxins form a pentameric arrangement which is responsible for binding to the receptor GM1 of the target epithelial cells of the host. Blocking these B pentamer-receptor interactions forms an avenue for therapeutic intervention. Here, the structural characterization of potential receptor-blocking compounds are described based on the previously identified inhibitor m-nitrophenyl-alpha-D-galactoside (MNPG). The structure of a CTB-MNPG complex confirms that the binding mode of this inhibitor is identical in the two homologous toxins CT and LT and is characterized by a glycosyl linkage geometry that leads to displacement of a well ordered water molecule near the amide group of Gly33 by the O1-substituent of MNPG. This glycosyl geometry is not maintained in the absence of a substituent that can displace this water, as shown by a complex of LTB with p-aminophenyl-alpha-D-galactoside (PAPG). New compounds were synthesized to investigate the feasibility of maintaining the favorable binding interactions exhibited by MNPG while gaining increased affinity through the addition of hydrophobic substituents complementary to either of two hydrophobic regions of the receptor-binding site. The structural characterization of complexes of LTB with two of these compounds, 3-benzylaminocarbonylphenyl-alpha-D-galactoside (BAPG) and 2-phenethyl-7-(2,3-dihydrophthalazine-1,4-dione)-alpha-D-galactoside (PEPG), demonstrates a partial success in this goal. Both compounds exhibit a mixture of binding modes, some of which are presumably influenced by the local packing environment at multiple crystallographically independent binding sites. The terminal phenyl ring of BAPG associates either with the phenyl group of Tyr12 or with the hydrophobic patch formed by Lys34 and Ile58. The latter interaction is also made by the terminal phenyl substituent of PEPG, despite a larger ring system linking the galactose moiety to the terminal phenyl. However, neither BAPG nor PEPG displaces the intended target water molecule. Both of the designed compounds exhibit increased affinity relative to the galactose and to PAPG notwithstanding the failure to displace a bound water, confirming that additional favorable hydrophobic interactions can be gained by extending the starting inhibitor by a hydrophobic tail. The insight gained from these structures should allow the design of additional candidate inhibitors that retain both the glycosyl geometry and water displacement exhibited by MNPG and the favorable hydrophobic interactions exhibited by BAPG and PEPG.