Biphenyl synthase,a novel type III polyketide synthase

Biphenyls and dibenzofurans are the phytoalexins of the Maloideae, a subfamily of the economically important Rosaceae. The carbon skeleton of the two classes of antimicrobial secondary metabolites is formed by biphenyl synthase (BIS). A cDNA encoding this key enzyme was cloned from yeast-extract-treated cell cultures of Sorbus aucuparia. BIS is a novel type III polyketide synthase (PKS) that shares about 60% amino acid sequence identity with other members of the enzyme superfamily. Its preferred starter substrate is benzoyl-CoA that undergoes iterative condensation with three molecules of malonyl-CoA to give 3,5-dihydroxybiphenyl via intramolecular aldol condensation. BIS did not accept CoA-linked cinnamic acids such as 4-coumaroyl-CoA. This substrate, however, was the preferential starter molecule for chalcone synthase (CHS) that was also cloned from S. aucuparia cell cultures. While BIS expression was rapidly, strongly and transiently induced by yeast extract treatment, CHS expression was not. In a phylogenetic tree, BIS grouped together closely with benzophenone synthase (BPS) that also uses benzoyl-CoA as starter molecule but cyclizes the common intermediate via intramolecular Claisen condensation. The molecular characterization of BIS thus contributes to the understanding of the functional diversity and evolution of type III PKSs.     

Biphenyl/diphenyl ether renin inhibitors: filling the S1 pocket of renin via the S3 pocket

Structure-based design led to the discovery of a novel class of renin inhibitors in which an unprecedented phenyl ring filling the S1 site is attached to the phenyl ring filling the S3 pocket. Optimization for several parameters including potency in the presence of human plasma, selectivity against CYP3A4 inhibition and improved rat oral bioavailability led to the identification of 8d which demonstrated antihypertensive efficacy in a transgenic rat model of human hypertension.     

Biotransformation of biphenyl by the filamentous fungus <Emphasis Type="Italic">Talaromyces helicus</Emphasis>

The filamentous fungusTalaromyces helicus , isolated from oil-contaminated sludge, oxidizes biphenyl via 4-hydroxybiphenyl to the dihydroxylated derivatives 4,4-dihydroxybiphenyl and 3,4-dihydroxybiphenyl, which, to a certain extent, are converted to glycosyl conjugates. The sugar moiety of the conjugate formed from 4,4-dihydroxybiphenyl was identified as glucose. Further metabolites: 2-hydroxybiphenyl, 2,5-dihydroxylated biphenyl, and the ring cleavage product 4-phenyl-2-pyrone-6-carboxylic acid accumulated only in traces. From these results the main pathway for biotransformation of biphenyl in T. helicus could be proposed to be the excretion of dihydroxylated derivatives (>75%) and their glucosyl conjugates (<25%).     

Catabolic role of a three-component salicylate oxygenase from Sphingomonas yanoikuyae B1 in polycyclic aromatic hydrocarbon degradation

Sphingomonas yanoikuyae B1 possesses several different multicomponent oxygenases involved in metabolizing aromatic compounds. Six different pairs of genes encoding large and small subunits of oxygenase iron-sulfur protein components have previously been identified in a gene cluster involved in the degradation of both monocyclic and polycyclic aromatic hydrocarbons. Insertional inactivation of one of the oxygenase large subunit genes, bphA1c, results in a mutant strain unable to grow on naphthalene, phenanthrene, or salicylate. The knockout mutant accumulates salicylate from naphthalene and 1-hydroxy-2-naphthoic acid from phenanthrene indicating the loss of salicylate oxygenase activity. Complementation experiments verify that the salicylate oxygenase in S. yanoikuyae B1 is a three-component enzyme consisting of an oxygenase encoded by bphA2cA1c, a ferredoxin encoded by the adjacent bphA3, and a ferredoxin reductase encoded by bphA4 located over 25kb away. Expression of bphA3-bphA2c-bphA1c genes in Escherichia coli demonstrated the ability of salicylate oxygenase to convert salicylate to catechol and 3-, 4-, and 5-methylsalicylate to methylcatechols.     

Fibrillogenesis in ADan peptides is inhibited by biphenyl ethers

In this study, biphenyl ethers of diverse functionality were used to assess their effect on fibrillogenesis of both the oxidized and reduced ADan peptides, in vitro. It was noted that these compounds not only stalled fibrillogenesis but were also able to disrupt pre-formed fibers. The EC₅₀ values for the inhibition of this process lie in the nanomolar range for 50 μM of peptide concentration, indicating the high potency of these compounds as inhibitors. It was found that these compounds impart to the peptides, an α-helical conformation which does not allow them to aggregate and form fibrils. These studies also point out that the transition of peptides through α-helical conformation may be a prelude to the onset of fibrillogenesis for oxADan peptides.     

Biphenyl amide p38 kinase inhibitors 3: Improvement of cellular and in vivo activity

The biphenyl amides (BPAs) are a novel series of p38α MAP kinase inhibitor. The optimisation of the series to give compounds that are potent in an in vivo disease model is discussed. SAR is presented and rationalised with reference to the crystallographic binding mode.     

Cyclomaltoheptaose mixed esters of anti-inflammatory drugs and short-chain fatty acids and study of their enzymatic hydrolysis in vitro

In an effort to enhance the drug-loading capacity of cyclomaltoheptaose (β-cyclodextrin, βCD) and to combine the function of anti-inflammatory drugs with short-chain fatty acids (SCFA), ternary esters incorporating seven copies of an anti-inflammatory drug and 14 copies of a SCFA onto a β-cyclodextrin core were designed and prepared. Acetic, propionic, or butyric esters were introduced at secondary OH groups, and ibuprofen, flurbiprofen, or felbinac was attached to primary OH groups through ester bonds. Heptakis[2,3-di-O-butanoyl-6-O-2-(biphenyl-4-yl)-ethanoyl]-cyclomaltoheptaose was very stable in aqueous and esterase solution. It was hydrolyzed by α-amylase (4 units/mL) with t_1/2 value of 18 h. The total released amount of biphenyl acetic acid was 38% after 24 h when the esterase was added after the α-amylase hydrolysis. The present results suggest that these nine βCD conjugates may release the anti-inflammatory drug in the colonic contents.     

Retuning Rieske-type oxygenases to expand substrate range

Rieske-type oxygenases are promising biocatalysts for the destruction of persistent pollutants or for the synthesis of fine chemicals. In this work, we explored pathways through which Rieske-type oxygenases evolve to expand their substrate range. BphAE(p4), a variant biphenyl dioxygenase generated from Burkholderia xenovorans LB400 BphAE(LB400) by the double substitution T335A/F336M, and BphAE(RR41), obtained by changing Asn(338), Ile(341), and Leu(409) of BphAE(p4) to Gln(338), Val(341), and Phe(409), metabolize dibenzofuran two and three times faster than BphAE(LB400), respectively. Steady-state kinetic measurements of single- and multiple-substitution mutants of BphAE(LB400) showed that the single T335A and the double N338Q/L409F substitutions contribute significantly to enhanced catalytic activity toward dibenzofuran. Analysis of crystal structures showed that the T335A substitution relieves constraints on a segment lining the catalytic cavity, allowing a significant displacement in response to dibenzofuran binding. The combined N338Q/L409F substitutions alter substrate-induced conformational changes of protein groups involved in subunit assembly and in the chemical steps of the reaction. This suggests a responsive induced fit mechanism that retunes the alignment of protein atoms involved in the chemical steps of the reaction. These enzymes can thus expand their substrate range through mutations that alter the constraints or plasticity of the catalytic cavity to accommodate new substrates or that alter the induced fit mechanism required to achieve proper alignment of reaction-critical atoms or groups.     

Degradation of chlorobiphenyls catalyzed by the bph-encoded biphenyl-2,3-dioxygenase and biphenyl-2,3-dihydrodiol-2,3-dehydrogenase of Pseudomonas sp. LB400

Abstract In order to characterize the metabolites produced in vivo by biphenyl-2,3-dioxygenase and biphenyl-2,3-dihydrodiol-2,3-dehydrogenase, the first two enzymes of the (polychloro)biphenyl catabolic pathway encoded by the bph locus of Pseudomonas sp. LB400, recombinant E. coli strains expressing the respective genes were constructed. Biphenyl-2,3-dioxygenase attack on 2,2'- or 2,4'-dichlorobiphenyl was shown to give rise to virtually quantitative ortho -dechlorination of these congeners by hydroxylation at the chlorinated carbon 2 and its unsubstituted neighbour. Elimination of hydrochloric acid directly leads to 2,3-dihydroxy-chlorobiphenyls and obviates the need for biphenyl-2,3-dihydrodiol-2,3-dehydrogenase for the catabolism of such congeners.     

Biphenyl sulfonic acid ligands for catalytic C-N cross coupling of aryl halides with anilines and secondary amines

The use of two biphenyl sulfonic acid ligands for the catalytic C-N cross coupling of aryl halides with anilines, 3-aminopyridine, and secondary amines is reported. Our results represent a significant improvement compared to state of the art methods especially with regards to the removal of palladium.