Crystal Structures of Acyl Carrier Protein in Complex with Two Catalytic Partners Show a Dynamic Role in Cellular Metabolism

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/107352/1/1079_ftp.pd

Control of Stereoselectivity in Diverse Hapalindole Metabolites is Mediated by Cofactor‐Induced Combinatorial Pairing of Stig Cyclases

Stereospecific polycyclic core formation of hapalindoles and fischerindoles is controlled by Stig cyclases through a three‐step cascade involving Cope rearrangement, 6‐exo‐trig cyclization, and a final electrophilic aromatic substitution. Reported here is a comprehensive study of all currently annotated Stig cyclases, revealing that these proteins can assemble into heteromeric complexes, induced by Ca2+, to cooperatively control the stereochemistry of hapalindole natural products.Die stereospezifische Bildung des polycyclischen Kerns der Hapalindole und Fischerindole wird durch Stig‐Cyclasen gesteuert, die eine dreistufige Kaskade aus Cope‐Umlagerung, 6‐exo‐trig‐Cyclisierung und elektrophiler aromatischer Substitution vermitteln. Die Proteine können sich induziert durch Ca2+ zu heterotrimeren Komplexen zusammenlagern, um auf kooperative Weise die Stereochemie zu steuern.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/155506/1/ange201913686.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/155506/2/ange201913686-sup-0001-misc_information.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/155506/3/ange201913686_am.pd

Structural and stereochemical diversity in prenylated indole alkaloids containing the bicyclo[2.2.2]diazaoctane ring system from marine and terrestrial fungi

Various fungi of the generaAspergillus,Penicillium, andMalbrancheaproduce prenylated indole alkaloids possessing a bicyclo[2.2.2]diazaoctane ring system.</p

Mechanism of double-base lesion bypass catalyzed by a Y-family DNA polymerase

As a widely used anticancer drug, cis-diamminedichloroplatinum(II) (cisplatin) reacts with adjacent purine bases in DNA to form predominantly cis-[Pt(NH3)2{d(GpG)-N7(1),-N7(2)}] intrastrand cross-links. Drug resistance, one of the major limitations of cisplatin therapy, is partially due to the inherent ability of human Y-family DNA polymerases to perform translesion synthesis in the presence of DNA-distorting damage such as cisplatin–DNA adducts. To better understand the mechanistic basis of translesion synthesis contributing to cisplatin resistance, this study investigated the bypass of a single, site-specifically placed cisplatin-d(GpG) adduct by a model Y-family DNA polymerase, Sulfolobus solfataricus DNA polymerase IV (Dpo4). Dpo4 was able to bypass this double-base lesion, although, the incorporation efficiency of dCTP opposite the first and second cross-linked guanine bases was decreased by 72- and 860-fold, respectively. Moreover, the fidelity at the lesion decreased up to two orders of magnitude. The cisplatin-d(GpG) adduct affected six downstream nucleotide incorporations, but interestingly the fidelity was essentially unaltered. Biphasic kinetic analysis supported a universal kinetic mechanism for the bypass of DNA lesions catalyzed by various translesion DNA polymerases. In conclusion, if human Y-family DNA polymerases adhere to this bypass mechanism, then translesion synthesis by these error-prone enzymes is likely accountable for cisplatin resistance observed in cancer patients

Structural Insights into the Function of the Nicotinate Mononucleotide:phenol/<i>p</i>‑cresol Phosphoribosyltransferase (ArsAB) Enzyme from <i>Sporomusa ovata</i>

Cobamides (Cbas) are cobalt (Co) containing tetrapyrrole-derivatives involved in enzyme-catalyzed carbon skeleton rearrangements, methyl-group transfers, and reductive dehalogenation. The biosynthesis of cobamides is complex and is only performed by some bacteria and achaea. Cobamides have an upper (<i>Coβ</i>) ligand (5′-deoxyadenosyl or methyl) and a lower (<i>Coα</i>) ligand base that contribute to the axial Co coordinations. The identity of the lower <i>Coα</i> ligand varies depending on the organism synthesizing the Cbas. The homoacetogenic bacterium <i>Sporomusa ovata</i> synthesizes two unique phenolic cobamides (i.e., Coα-(phenolyl/<i>p</i>-cresolyl)­cobamide), which are used in the catabolism of methanol and 3,4-dimethoxybenzoate by this bacterium. The <i>S. ovata</i> ArsAB enzyme activates a phenolic lower ligand prior to its incorporation into the cobamide. ArsAB consists of two subunits, both of which are homologous (∼35% identity) to the well-characterized <i>Salmonella enterica</i> CobT enzyme, which transfers nitrogenous bases such as 5,6-dimethylbenzimidazole (DMB) and adenine, but cannot utilize phenolics. Here we report the three-dimensional structure of ArsAB, which shows that the enzyme forms a pseudosymmetric heterodimer, provide evidence that only the ArsA subunit has base:phosphoribosyl-transferase activity, and propose a mechanism by which phenolic transfer is facilitated by an activated water molecule

Bioprospecting for Trichothecene 3-O-Acetyltransferases in the Fungal Genus Fusarium Yields Functional Enzymes with Different Abilities To Modify the Mycotoxin Deoxynivalenol▿ †

The trichothecene mycotoxin deoxynivalenol (DON) is a common contaminant of small grains, such as wheat and barley, in the United States. New strategies to mitigate the threat of DON need to be developed and implemented. TRI101 and TRI201 are trichothecene 3-O-acetyltransferases that are able to modify DON and reduce its toxicity. Recent work has highlighted differences in the activities of TRI101 from two different species of Fusarium (F. graminearum and F. sporotrichioides), but little is known about the relative activities of TRI101/TRI201 enzymes produced by other species of Fusarium. We cloned TRI101 or TRI201 genes from seven different species of Fusarium and found genetic identity between sequences ranging from 66% to 98%. In vitro feeding studies using transformed yeast showed that all of the TRI101/TRI201 enzymes tested were able to acetylate DON; conversion of DON to 3-acetyl-deoxynivalenol (3ADON) ranged from 50.5% to 100.0%, depending on the Fusarium species from which the gene originated. A time course assay showed that the rate of acetylation varied from species to species, with the gene from F. sporotrichioides having the lowest rate. Steady-state kinetic assays using seven purified enzymes produced catalytic efficiencies for DON acetylation ranging from 6.8 × 104 M−1·s−1 to 4.7 × 106 M−1·s−1. Thermostability measurements for the seven orthologs ranged from 37.1°C to 43.2°C. Extended sequence analysis of portions of TRI101/TRI201 from 31 species of Fusarium (including known trichothecene producers and nonproducers) suggested that other members of the genus may contain functional TRI101/TRI201 genes, some with the potential to outperform those evaluated in the present study

Structural Insights into the Mechanism of Four-Coordinate Cob(II)alamin Formation in the Active Site of the <i>Salmonella enterica</i> ATP:Co(I)rrinoid Adenosyltransferase Enzyme: Critical Role of Residues Phe91 and Trp93

ATP:co­(I)­rrinoid adenosyltransferases (ACATs) are enzymes that catalyze the formation of adenosylcobalamin (AdoCbl, coenzyme B₁₂) from cobalamin and ATP. There are three families of ACATs, namely, CobA, EutT, and PduO. In <i>Salmonella enterica</i>, CobA is the housekeeping enzyme that is required for de novo AdoCbl synthesis and for salvaging incomplete precursors and cobalamin from the environment. Here, we report the crystal structure of CobA in complex with ATP, four-coordinate cobalamin, and five-coordinate cobalamin. This provides the first crystallographic evidence of the existence of cob­(II)­alamin in the active site of CobA. The structure suggests a mechanism in which the enzyme adopts a closed conformation and two residues, Phe91 and Trp93, displace 5,6-dimethylbenzimidazole, the lower nucleotide ligand base of cobalamin, to generate a transient four-coordinate cobalamin, which is critical in the formation of the AdoCbl Co–C bond. In vivo and in vitro mutational analyses of Phe91 and Trp93 emphasize the important role of bulky hydrophobic side chains in the active site. The proposed manner in which CobA increases the redox potential of the cob­(II)­alamin/cob­(I)­alamin couple to facilitate formation of the Co–C bond appears to be analogous to that utilized by the PduO-type ACATs, where in both cases the polar coordination of the lower ligand to the cobalt ion is eliminated by placing that face of the corrin ring adjacent to a cluster of bulky hydrophobic side chains