The C-terminal thioesterase domain of the nonribosomal peptide synthetase producing the lipopetide surfactin (Srf TE) retains autonomous ability to generate the cyclic peptidolactone skeleton of surfactin when provided with a soluble beta-hydroxy-butyryl-heptapeptidyl thioester substrate. Utilizing the recently solved crystal structure [Bruner, S. D., et al. (2002) Structure 10, 301-310], the active-site nucleophile, Ser80, was changed to Cys, and the other members of the catalytic triad, Asp107 and His207, were changed to Ala, with the resulting mutants lacking detectable activity. Two cationic side chains in the active site, Lys111 and Arg120, were changed to Ala, causing an increased partitioning of the product to hydrolysis, as did a P26G mutant, mimicking the behavior of lipases. To evaluate recognition elements in substrates used by Srf TE, alterations to the fatty acyl group, the heptapeptide, and the thioester leaving group were made, and the resulting substrates were characterized for kinetic competency and flux of product to cyclization or hydrolysis. Alterations that could be accepted for cyclization were identified in all three parts of the substrate, although tolerance limits for changes varied. In addition, cocrystal structures of Srf TE with dipeptidyl boronate inhibitors were solved, illustrating the critical binding determinants of the substrate. On the basis of the structures and biochemical data, the cyclizing conformation of the surfactin peptide was modeled into the enzyme active site.

Four proteins, DpgA-D, required for the biosynthesis by actinomycetes of the nonproteinogenic amino acid monomer (S)-3,5-dihydroxyphenylglycine (Dpg), that is a crosslinking site in the maturation of vancomycin and teicoplanin antibiotic scaffolds, were expressed in Escherichia coli, purified in soluble form, and assayed for enzymatic activity. DpgA is a type III polyketide synthase, converting four molecules of malonyl-CoA to 3,5-dihydroxyphenylacetyl-CoA (DPACoA) and three free coenzyme A (CoASH) products. Almost no turnover was observed for DpgA until DpgB was added, producing a net k cat of 1-2 min ؊1 at a 3:1 ratio of DpgB:DpgA. Addition of DpgD gave a further 2-fold rate increase. DpgC had the unusual catalytic capacity to convert DPA-CoA to 3,5-dihydroxyphenylglyoxylate, which is a transamination away from Dpg. DpgC performed a net CH 2 to CAO four-electron oxidation on the C␣ of DPA-CoA and hydrolyzed the thioester linkage with a k cat of 10 min ؊1 . Phenylacetyl-CoA was also processed, to phenylglyoxylate, but with about 500-fold lower k cat͞KM. DpgC showed no activity in anaerobic incubations, suggesting an oxygenase function, but had no detectable bound organic cofactors or metals. A weak enoyl-CoA hydratase activity was detected for both DpgB and DpgD.

3,5-Dihydroxyphenylglycine is a crucial amino acid monomer in the nonribosomal glycopeptide antibiotic vancomycin. This nonproteinogenic amino acid is constructed from malonyl-CoA by a set of four enzymes, DpgA-D, in the biosynthetic cluster. DpgC is an unusual metal-free, cofactor-free enzyme that consumes O(2) during the conversion of 3,5-dihydroxyphenylacetyl-CoA (DPA-CoA) to the penultimate intermediate 3,5-dihydroxyphenylglyoxylate (DPGx). We show that in anaerobic incubations, DpgC catalyzes the exchange of the C(2)-methylene hydrogens of DPA-CoA at unequal rates, consistent with enzyme-mediated formation of the substrate-derived C(2)-carbanion as an early intermediate. Incubations with (18)O(2) reveal that DpgC transfers both atoms of an O(2) molecule to DPGx product. This establishes DpgC as a 1,2-dioxygenase that mediates thioester cleavage by the oxygen transfer process. These results are consistent with a DPA-CoA C(2)-peroxy intermediate, followed by enzyme-directed alpha-peroxylactone formation and collapse by O-O bond cleavage.

DpgA is a bacterial type III polyketide synthase (PKS) that decarboxylates and condenses four malonyl-CoA molecules to produce 3,5-dihydroxyphenylacetyl-CoA (DPA-CoA) in the biosynthetic pathway to 3,5-dihydroxyphenylglycine, a key nonproteinogenic residue in the vancomycin family of antibiotics. DpgA has the conserved catalytic triad of Cys/His/Asn typical of type III PKS enzymes, and has been assumed to use Cys160 as the catalytic nucleophile to create a series of elongating acyl-S-enzyme intermediates prior to the C(8) to C(3) cyclization step. Incubation of purified DpgA with [(14)C]-malonyl-CoA followed by acid quench during turnover leads to accumulation of 10-15% of the DpgA molecules covalently acylated. Mutation of the active site Cys160 to Ala abrogated detectable covalent acylation, but the C160A mutant retained 50% of the V(max) for DPA-CoA formation, with a k(cat) still at 0.5 catalytic turnovers/min. For comparison, a C190A mutant retained wild-type activity, while the H296A mutant, in which the side chain of the presumed catalytic His is removed, had a 6-fold drop in k(cat). During turnover, purified DpgA produced 1.2 equivalents of acetyl-CoA for each DPA-CoA, indicating 23% uncoupled decarboxylation competing with condensative C-C coupling. The C160A mutant showed an increased partition ratio for malonyl-CoA decarboxylation to acetyl-CoA vs condensation to DPA-CoA, reflecting more uncoupling in the mutant enzyme. The Cys-to-Ala mutant thus shows the unexpected result that, when the normal acyl-S-enzyme mechanism for this type III PKS elongation/cyclization catalyst is removed, it can still carry out the regioselective construction of the eight-carbon DPA-CoA skeleton with surprising efficiency.

Context plays an important role in visual recognition. Recent studies have shown that visual recognition networks can be fooled by placing objects in inconsistent contexts (e.g. a cow in the ocean). To understand and model the role of contextual information in visual recognition, we systematically and quantitatively investigated ten critical properties of where, when, and how context modulates recognition including amount of context, context and object resolution, geometrical structure of context, context congruence, time required to incorporate contextual information, and temporal dynamics of contextual modulation. The tasks involve recognizing a target object surrounded with context in a natural image. As an essential benchmark, we first describe a series of psychophysics experiments, where we alter one aspect of context at a time, and quantify human recognition accuracy. To computationally assess performance on the same tasks, we propose a biologically inspired context aware object recognition model consisting of a two-stream architecture. The model processes visual information at the fovea and periphery in parallel, dynamically incorporates both object and contextual information, and sequentially reasons about the class label for the target object. Across a wide range of behavioral tasks, the model approximates human level performance without retraining for each task, captures the dependence of context enhancement on image properties, and provides initial steps towards integrating scene and object information for visual recognition.

Figure 1: Images under normal context and out-of-context conditions were generated in the VirtualHome environment [28] using the Unity 3D simulation engine [20]. One set of examples is shown. The same target object (a mug indicated by the red bounding box) is shown in different context conditions: normal context and out-of-context conditions including gravity (target object is floating in the air), object co-occurrence statistics, combination of both gravity and object co-occurrence statistics, enlarged object size, and no context with uniform grey pixels as background.

The 207-kDa polyketide synthase (PKS) module (residues 1-1895) and the 143-kDa nonribosomal peptidyl synthetase (NRPS) module (1896 -3163) of the 350-kDa HMWP1 subunit of yersiniabactin synthetase have been expressed in and purified from Escherichia coli in soluble forms to characterize the acyl carrier protein (ACP) domain of the PKS module and the homologous peptidyl carrier protein (PCP3) domain of the NRPS module. The apo-ACP and PCP domains could be selectively posttranslationally primed by the E. coli ACPS and EntD phosphopantetheinyl transferases (PPTases), respectively, whereas the Bacillus subtilis PPTase Sfp primed both carrier protein domains in vitro or during in vivo coexpression. The holo-NRPS module but not the holo-PKS module was then selectively aminoacylated with cysteine by the adenylation domain embedded in the HMWP2 subunit of yersiniabactin synthetase, acting in trans. When the acyltransferase (AT) domain of HMWP1 was analyzed for its ability to malonylate the holo carrier protein domains, in cis acylation was first detected. Then, in trans malonylation of the excised holo-ACP or holo-PCP3-TE fragments by HMWP1 showed both were malonylated with a 3:1 catalytic efficiency ratio, showing a promiscuity to the AT domain. Y ersiniabactin (Ybt) is an iron-chelating siderophore produced by the plague pathogen Yersinia pestis in irondeficient environments and serves as a virulence factor to permit these disease-causing bacteria to grow effectively in animals and humans (1-4). Ybt is assembled by a three-subunit system, YbtE, HMWP2, and HMWP1 (Fig. 1A), comprising 17 predicted domains that function as a mixed nonribosomal peptidyl synthetase (NRPS)-polyketide synthase (PKS)-nonribosomal peptidyl synthetase assembly line (5). The four rings of Ybt (Fig. 1B) are derived from salicylate and three cysteines (5-8) with a malonyl CoA-derived t-butyl linker between the second and third five-membered heterocycles. In the 17 predicted domains of Ybt synthetase are five carrier proteins, three in HMWP2 and two in HMWP1, all of which have been shown (refs. 5, 6, 9, and this work) to be primed by phosphopantetheinylation and serve as the sites for acylation by salicyl (ArCP), cysteinyl [peptidyl carrier protein (PCP) 1 , PCP 2 , PCP 3 ], or malonyl (acyl carrier protein, ACP) groups in Ybt chain growth.Yersiniabactin is an example of a mixed nonribosomal peptide and polyketide, as are the natural products bleomycin (10), epothilone (11, 12), rapamycin (13), and FK506 (14). The molecular intersections of the NRPS and PKS assembly lines to produce these mixed products are of particular interest for both fundamental enzymology of natural product biosynthesis and for design and execution of combinatorial biosynthesis strategies. The HMWP1 subunit, 350 kDa with 9 predicted domains (5, 15) ( Fig. 1 A), contains a predicted PKS module, ketoacyl synthase (KS), acyltransferase (AT), methyltransferase (MT 2 ), ketoacyl reductase (KR), and ACP in the first 207 kDa, followed by a 4-domain 143-kDa NRPS module, condensation͞...

Context plays an important role in visual recognition. Recent studies have shown that visual recognition networks can be fooled by placing objects in inconsistent contexts (e.g. a cow in the ocean). To understand and model the role of contextual information in visual recognition, we systematically and quantitatively investigated ten critical properties of where, when, and how context modulates recognition including amount of context, context and object resolution, geometrical structure of context, context congruence, time required to incorporate contextual information, and temporal dynamics of contextual modulation. The tasks involve recognizing a target object surrounded with context in a natural image. As an essential benchmark, we first describe a series of psychophysics experiments, where we alter one aspect of context at a time, and quantify human recognition accuracy. To computationally assess performance on the same tasks, we propose a biologically inspired context aware object recognition model consisting of a two-stream architecture. The model processes visual information at the fovea and periphery in parallel, dynamically incorporates both object and contextual information, and sequentially reasons about the class label for the target object. Across a wide range of behavioral tasks, the model approximates human level performance without retraining for each task, captures the dependence of context enhancement on image properties, and provides initial steps towards integrating scene and object information for visual recognition.