Brian D Ames

Harvard Medical School, Boston, Massachusetts, United States

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Publications (16)93.17 Total impact

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    ABSTRACT: Aerial spores, crucial for propagation and dispersal of the Kingdom Fungi, are commonly the initial inoculum of pathogenic fungi. Natural products (secondary metabolites) have been correlated with fungal spore development and enhanced virulence in the human pathogen Aspergillus fumigatus but mechanisms for metabolite deposition in the spore are unknown. Metabolomic profiling of A. fumigatus deletion mutants of fumiquinazoline (Fq) cluster genes reveal that the first two products of the Fq cluster, FqF and FqA, are produced to comparable levels in all fungal tissues but the final enzymatically-derived product, FqC, predominantly accumulates in the fungal spore. Loss of the sporulation-specific transcription factor, BrlA, yields a strain unable to produce FqA or FqC. Fluorescence microscopy showed FmqD, the oxidoreductase required to generate FqC, was secreted via the Golgi apparatus to the cell wall in an actin-dependent manner. In contrast, all other members of the Fq pathway including the putative transporter, FmqE - which had no effect on Fq biosynthesis - were internal to the hyphae. The coordination of BrlA-mediated tissue specificity with FmqD secretion to the cell wall presents a previously undescribed mechanism to direct localization of specific secondary metabolites to spores of the differentiating fungus.
    Cellular Microbiology 02/2014; · 4.81 Impact Factor
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    ABSTRACT: Complexity generation in naturally occurring peptide scaffolds can occur either by posttranslational modifications of nascent ribosomal proteins or through post assembly line tailoring of nonribosomal peptides. Short enzymatic pathways utilizing bimodular and trimodular nonribosomal peptide synthetase (NRPS) assembly lines, followed by tailoring acyltransferases, oxygenases and/or prenyltransferases, efficiently construct complex fungal peptidyl alkaloid scaffolds in Aspergilli, Neosartorya and Penicillium species. Use of the nonproteinogenic amino acid anthranilate both as chain-initiating building block and chain-terminating intramolecular nucleophile efficiently leads to a variety of peptidyl alkaloid scaffolds with two to seven fused rings.
    ACS Chemical Biology 05/2013; · 5.44 Impact Factor
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    ABSTRACT: Cyclization of linear peptidyl precursors produced by nonribosomal peptide synthetases (NRPSs) is an important step in the biosynthesis of bioactive cyclic peptides. Whereas bacterial NRPSs use thioesterase domains to perform the cyclization, fungal NRPSs have apparently evolved to use a different enzymatic route. In verified fungal NRPSs that produce macrocyclic peptides, each megasynthetase terminates with a condensation-like (C(T)) domain that may perform the macrocyclization reaction. To probe the role of such a C(T) domain, we reconstituted the activities of the Penicillium aethiopicum trimodular NPRS TqaA in Saccharomyces cerevisiae and in vitro. Together with the reconstituted bimodular NRPS AnaPS, we dissected the cyclization steps of TqaA in transforming the linear anthranilate-D-tryptophan-L-alanyl tripeptide into fumiquinazoline F. Extensive biochemical and mutational studies confirmed the essential role of the C(T) domain in catalyzing cyclization in a thiolation domain-dependent fashion. Our work provides evidence of a likely universal macrocyclization strategy used by fungal NRPSs.
    Nature Chemical Biology 08/2012; · 12.95 Impact Factor
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    ABSTRACT: Lovastatin is an important statin prescribed for the treatment and prevention of cardiovascular diseases. Biosynthesis of lovastatin uses an iterative type I polyketide synthase (PKS). LovC is a trans-acting enoyl reductase (ER) that specifically reduces three out of eight possible polyketide intermediates during lovastatin biosynthesis. Such trans-acting ERs have been reported across a variety of other fungal PKS enzymes as a strategy in nature to diversify polyketides. How LovC achieves such specificity is unknown. The 1.9-Å structure of LovC reveals that LovC possesses a medium-chain dehydrogenase/reductase (MDR) fold with a unique monomeric assembly. Two LovC cocrystal structures and enzymological studies help elucidate the molecular basis of LovC specificity, define stereochemistry, and identify active-site residues. Sequence alignment indicates a general applicability to trans-acting ERs of fungal PKSs, as well as their potential application to directing biosynthesis.
    Proceedings of the National Academy of Sciences 06/2012; 109(28):11144-9. · 9.81 Impact Factor
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    ABSTRACT: Aromatic polyketides are biologically active natural products. Many important pharmaceuticals are derived from aromatic polyketides. Especially important in aromatic polyketide biosynthesis is the regiospecific cyclization of a linear, preassembled polyketide chain catalyzed by aromatase/cyclase (ARO/CYC), which serves as a key control point in aromatic ring formation. How different ARO/CYCs promote different cyclization patterns is not well understood. The whiE locus of Streptomyces coelicolor A3(2) is responsible for the biosynthesis of an aromatic polyketide precursor to the gray spore pigment. The WhiE ARO/CYC catalyzes the regiospecific C9-C14 and C7-C16 cyclization and aromatization of a 24-carbon polyketide chain. WhiE ARO/CYC shares a high degree of similarity to another nonreducing PKS ARO/CYC, TcmN ARO/CYC. This paper presents the apo crystal structure of WhiE ARO/CYC, and cocrystal structures of WhiE and TcmN ARO/CYCs bound with polycyclic aromatic compounds that mimic the respective ARO/CYC products. Site-directed mutagenesis coupled with in vitro PKS reconstitution assays was used to characterize the interior pocket residues of WhiE ARO/CYC. The results confirmed that the interior pocket of ARO/CYCs is a critical determinant of polyketide cyclization specificity. A unified ARO/CYC-mediated cyclization mechanism is proposed on the basis of these structural and functional results.
    Biochemistry 03/2012; 51(14):3079-91. · 3.38 Impact Factor
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    ABSTRACT: The ortho-, meta-, and para- regioisomers of aminobenzoate are building blocks for a wide range of microbial natural products. Both the ortho-isomer (anthranilate) and PABA derive from the central shikimate pathway metabolite chorismate while the meta-isomer is not available by that route and starts from UDP-3-aminoglucose. PABA is largely funnelled into folate biosynthesis while anthranilate is the scaffold for biosynthetic elaboration into many natural heterocycles, most notably with its role in indole formation for tryptophan biosynthesis. Anthranilate is also converted to benzodiazepinones, fumiquinazolines, quinoxalines, phenoxazines, benzoxazolinates, quinolones, and phenazines, often with redox enzyme participation. The 5-hydroxy form of 3-aminobenzaote is the starter unit for ansa-bridged rifamycins, ansamitocins, and geldanamycins, whereas regioisomers 2-hydroxy, 4-hydroxy and 2,4-dihydroxy-3-aminobenzoate are key components of antimycin, grixazone, and platencin and platensimycin biosynthesis, respectively. The enzymatic mechanisms for generation of the aminobenzoate regioisomers and their subsequent utilization for diverse heterocycle and macrocycle construction are examined.
    Natural Product Reports 11/2011; 29(1):37-59. · 10.18 Impact Factor
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    ABSTRACT: The human pathogen Aspergillus fumigatus makes a series of fumiquinazoline (FQ) peptidyl alkaloids of increasing scaffold complexity using L-Trp, 2 equiv of L-Ala, and the non-proteinogenic amino acid anthranilate as building blocks. The FQ gene cluster encodes two non-ribosomal peptide synthetases (NRPS) and two flavoproteins. The trimodular NRPS Af12080 assembles FQF (the first level of complexity) while the next two enzymes, Af12060 and Af12050, act in tandem in an oxidative annulation sequence to couple alanine to the indole side chain of FQF to yield the imidazolindolone-containing FQA. In this study we show that the fourth enzyme, the monocovalent flavoprotein Af12070, introduces a third layer of scaffold complexity by converting FQA to the spirohemiaminal FQC, presumably by catalyzing the formation of a transient imine within the pyrazinone ring (and therefore acting in an unprecedented manner as an FAD-dependent amide oxidase). FQC subsequently converts nonenzymatically to the known cyclic aminal FQD. We also investigated the effect of substrate structure on Af12070 activity and subsequent cyclization with a variety of FQA analogues, including an FQA diastereomer (2'-epi-FQA), which is an intermediate in the fungal biosynthesis of the tremorgenic tryptoquialanine. 2'-epi-FQA is processed by Af12070 to epi-FQD, not epi-FQC, illustrating that the delicate balance in product cyclization regiochemistry can be perturbed by a remote stereochemical center.
    Biochemistry 09/2011; 50(40):8756-69. · 3.38 Impact Factor
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    ABSTRACT: Aromatic polyketides comprise an important class of natural products that possess a wide range of biological activities. The cyclization of the polyketide chain is a critical control point in the biosynthesis of aromatic polyketides. The aromatase/cyclases (ARO/CYCs) are an important component of the type II polyketide synthase (PKS) and help fold the polyketide for regiospecific cyclizations of the first ring and/or aromatization, promoting two commonly observed first-ring cyclization patterns for the bacterial type II PKSs: C7-C12 and C9-C14. We had previously reported the crystal structure and enzymological analyses of the TcmN ARO/CYC, which promotes C9-C14 first-ring cyclization. However, how C7-C12 first-ring cyclization is controlled remains unresolved. In this work, we present the 2.4 Å crystal structure of ZhuI, a C7-C12-specific first-ring ARO/CYC from the type II PKS pathway responsible for the production of the R1128 polyketides. Though ZhuI possesses a helix-grip fold shared by TcmN ARO/CYC, there are substantial differences in overall structure and pocket residue composition that may be important for directing C7-C12 (rather than C9-C14) cyclization. Docking studies and site-directed mutagenesis coupled to an in vitro activity assay demonstrate that ZhuI pocket residues R66, H109, and D146 are important for enzyme function. The ZhuI crystal structure helps visualize the structure and putative dehydratase function of the didomain ARO/CYCs from KR-containing type II PKSs. The sequence-structure-function analysis described for ZhuI elucidates the molecular mechanisms that control C7-C12 first-ring polyketide cyclization and builds a foundation for future endeavors into directing cyclization patterns for engineered biosynthesis of aromatic polyketides.
    Biochemistry 08/2011; 50(39):8392-406. · 3.38 Impact Factor
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    ABSTRACT: The fungal peptidyl alkaloids of the tryptoquialanine and fumiquinazoline families are nonribosomally assembled by annulation of the indole side chain of fumiquinazoline F (FQF) with an alaninyl or aminoisobutyryl unit by monomodular NRPS enzymes containing adenylation, thiolation, and condensation (A-T-C) domains. The Af12060 and Af12050 enzyme pair from Aspergillus fumigatus thereby converts FQF to FQA, while the homologous TqaH and TqaB enzyme pair from Penicillium aethiopicum makes the 2'-epi diastereomer of FQA, differing only in the stereochemistry of one of the C-N bonds formed in the annulation with l-Ala. To evaluate the basis for this stereochemical control, we have mixed and matched the flavoprotein oxygenases Af12060 and TqaH with the A-T-C modular enzymes Af12050 and TqaB to show that the NRPS enzymes control the stereochemical outcome. The terminal 50 kDa condensation domains of Af12050 and TqaB are solely responsible for the stereochemical control as shown both by making chimeric (e.g., A-T-C* and A*-T*-C) forms of these monomodular NRPS enzymes and by expression, purification, and assay of the excised C-domains. The Af12050 and TqaB condensation domains are thus a paired set of diastereospecific annulation catalysts that act on the fumiquinazoline F scaffold.
    Biochemistry 06/2011; 50(25):5668-79. · 3.38 Impact Factor
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    ABSTRACT: Phenylalanine hydroxylase (PheH) is an iron(II)-dependent enzyme that catalyzes the hydroxylation of aromatic amino acid l-phenylalanine (L-Phe) to l-tyrosine (L-Tyr). The enzymatic modification has been demonstrated to be highly regiospecific, forming proteinogenic para-Tyr (p-Tyr) exclusively. Here we biochemically characterized the first example of a phenylalanine 3-hydroxylase (Phe3H) that catalyzes the synthesis of meta-Tyr (m-Tyr) from Phe. Subsequent mutagenesis studies revealed that two residues in the active site of Phe3H (Cys187 and Thr202) contribute to C-3 rather than C-4 hydroxylation of the phenyl ring. This work sets the stage for the mechanistic and structural study of regiospecific control of the substrate hydroxylation by PheH.
    Biochemistry 06/2011; 50(24):5401-3. · 3.38 Impact Factor
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    ABSTRACT: Tremorgenic mycotoxins are a group of indole alkaloids which include the quinazoline-containing tryptoquivaline (2) that are capable of eliciting intermittent or sustained tremors in vertebrate animals. The biosynthesis of this group of bioactive compounds, which are characterized by an acetylated quinazoline ring connected to a 6-5-5 imidazoindolone ring system via a 5-membered spirolactone, has remained uncharacterized. Here, we report the identification of a gene cluster (tqa) from P. aethiopicum that is involved in the biosynthesis of tryptoquialanine (1), which is structurally similar to 2. The pathway has been confirmed to go through an intermediate common to the fumiquinazoline pathway, fumiquinazoline F, which originates from a fungal trimodular nonribosomal peptide synthetase (NRPS). By systematically inactivating every biosynthetic gene in the cluster, followed by isolation and characterization of the intermediates, we were able to establish the biosynthetic sequence of the pathway. An unusual oxidative opening of the pyrazinone ring by an FAD-dependent berberine bridge enzyme-like oxidoreductase has been proposed based on genetic knockout studies. Notably, a 2-aminoisobutyric acid (AIB)-utilizing NRPS module has been identified and reconstituted in vitro, along with two putative enzymes of unknown functions that are involved in the synthesis of the unnatural amino acid by genetic analysis. This work provides new genetic and biochemical insights into the biosynthesis of this group of fungal alkaloids, including the tremorgens related to 2.
    Journal of the American Chemical Society 02/2011; 133(8):2729-41. · 10.68 Impact Factor
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    ABSTRACT: Aspergillus fumigatus Af293 is a known producer of quinazoline natural products, including the antitumor fumiquinazolines, of which the simplest member is fumiquinazoline F (FQF) with a 6-6-6 tricyclic core derived from anthranilic acid, tryptophan, and alanine. FQF is the proposed biological precursor to fumiquinazoline A (FQA) in which the pendant indole side chain has been modified via oxidative coupling of an additional molecule of alanine, yielding a fused 6-5-5 imidazoindolone. We recently identified fungal anthranilate-activating nonribosomal peptide synthetase (NRPS) domains through bioinformatics approaches. One domain previously identified is part of the trimodular NRPS Af12080, which we predict is responsible for FQF formation. We now show that two adjacent A. fumigatus ORFs, a monomodular NRPS Af12050 and a flavoprotein Af12060, are necessary and sufficient to convert FQF to FQA. Af12060 oxidizes the 2',3'-double bond of the indole side chain of FQF, and the three-domain NRPS Af12050 activates l-Ala as the adenylate, installs it as the pantetheinyl thioester on its carrier protein domain, and acylates the oxidized indole for subsequent intramolecular cyclization to create the 6-5-5 imidazolindolone of FQA. This work provides experimental validation of the fumiquinazoline biosynthetic cluster of A. fumigatus Af293 and describes an oxidative annulation biosynthetic strategy likely shared among several classes of polycyclic fungal alkaloids.
    Biochemistry 10/2010; 49(39):8564-76. · 3.38 Impact Factor
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    Brian D Ames, Christopher T Walsh
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    ABSTRACT: Fungal natural products containing benzodiazepinone- and quinazolinone-fused ring systems can be assembled by nonribosomal peptide synthetases (NRPS) using the conformationally restricted beta-amino acid anthranilate as one of the key building blocks. We validated that the first module of the acetylaszonalenin synthetase of Neosartorya fischeri NRRL 181 activates anthranilate to anthranilyl-AMP. With this as a starting point, we then used bioinformatic predictions about fungal adenylation domain selectivities to identify and confirm an anthranilate-activating module in the fumiquinazoline A producer Aspergillus fumigatus Af293 as well as a second anthranilate-activating NRPS in N. fischeri. This establishes an anthranilate adenylation domain code for fungal NRPS and should facilitate detection and cloning of gene clusters for benzodiazepine- and quinazoline-containing polycyclic alkaloids with a wide range of biological activities.
    Biochemistry 03/2010; 49(15):3351-65. · 3.38 Impact Factor
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    Shiou-Chuan Sheryl Tsai, Brian Douglas Ames
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    ABSTRACT: This chapter describes structural and associated enzymological studies of polyketide synthases, including isolated single domains and multidomain fragments. The sequence-structure-function relationship of polyketide biosynthesis, compared with homologous fatty acid synthesis, is discussed in detail. Structural enzymology sheds light on sequence and structural motifs that are important for the precise timing, substrate recognition, enzyme catalysis, and protein-protein interactions leading to the extraordinary structural diversity of naturally occurring polyketides.
    Methods in enzymology 02/2009; 459:17-47. · 1.90 Impact Factor
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    ABSTRACT: Polyketides are a class of natural products with highly diverse chemical structures and pharmaceutical activities. Polyketide cyclization, promoted by the aromatase/cyclase (ARO/CYC), helps diversify aromatic polyketides. How the ARO/CYC promotes highly specific cyclization is not well understood because of the lack of a first-ring ARO/CYC structure. The 1.9 A crystal structure of Tcm ARO/CYC reveals that the enzyme belongs to the Bet v1-like superfamily (or STAR domain family) with a helix-grip fold, and contains a highly conserved interior pocket. Docking, mutagenesis, and an in vivo assay show that the size, shape, and composition of the pocket are important to orient and specifically fold the polyketide chain for C9-C14 first-ring and C7-C16 second-ring cyclizations. Two pocket residues, R69 and Y35, were found to be essential for promoting first- and second-ring cyclization specificity. Different pocket residue mutations affected the polyketide product distribution. A mechanism is proposed based on the structure-mutation-docking results. These results strongly suggest that the regiospecific cyclizations of the first two rings and subsequent aromatizations take place in the interior pocket. The chemical insights gleaned from this work pave the foundation toward defining the molecular rules for the ARO/CYC cyclization specificity, whose rational control will be important for future endeavors in the engineered biosynthesis of novel anticancer and antibiotic aromatic polyketides.
    Proceedings of the National Academy of Sciences 05/2008; 105(14):5349-54. · 9.81 Impact Factor
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    ABSTRACT: Tetracyclines are aromatic polyketides biosynthesized by bacterial type II polyketide synthases (PKSs). Understanding the biochemistry of tetracycline PKSs is an important step toward the rational and combinatorial manipulation of tetracycline biosynthesis. To this end, we have sequenced the gene cluster of oxytetracycline (oxy and otc genes) PKS genes from Streptomyces rimosus. Sequence analysis revealed a total of 21 genes between the otrA and otrB resistance genes. We hypothesized that an amidotransferase, OxyD, synthesizes the malonamate starter unit that is a universal building block for tetracycline compounds. In vivo reconstitution using strain CH999 revealed that the minimal PKS and OxyD are necessary and sufficient for the biosynthesis of amidated polyketides. A novel alkaloid (WJ35, or compound 2) was synthesized as the major product when the oxy-encoded minimal PKS, the C-9 ketoreductase (OxyJ), and OxyD were coexpressed in CH999. WJ35 is an isoquinolone compound derived from an amidated decaketide backbone and cyclized with novel regioselectivity. The expression of OxyD with a heterologous minimal PKS did not afford similarly amidated polyketides, suggesting that the oxy-encoded minimal PKS possesses novel starter unit specificity.
    Applied and Environmental Microbiology 05/2006; 72(4):2573-80. · 3.95 Impact Factor

Publication Stats

224 Citations
93.17 Total Impact Points

Institutions

  • 2010–2014
    • Harvard Medical School
      • Department of Biological Chemistry and Molecular Pharmacology
      Boston, Massachusetts, United States
  • 2008–2012
    • University of California, Irvine
      • • Department of Chemistry
      • • Department of Molecular Biology and Biochemistry
      Irvine, CA, United States
  • 2006–2012
    • University of California, Los Angeles
      • Department of Chemical and Biomolecular Engineering
      Los Angeles, CA, United States