ABSTRACTMany investigations have provided evidence that plant secondary metabolites, especially flavonoids, may serve as signal molecules to trigger the abilities of bacteria to degrade chlorobiphenyls in soil. However, the bases for this interaction are largely unknown. In this work, we found that BphAEB356, the biphenyl/chlorobiphenyl dioxygenase fromPandoraea pnomenusaB356, is significantly better fitted to metabolize flavone, isoflavone, and flavanone than BphAELB400fromBurkholderia xenovoransLB400. Unlike those of BphAELB400, the kinetic parameters of BphAEB356toward these flavonoids were in the same range as for biphenyl. In addition, remarkably, the biphenyl catabolic pathway of strain B356 was strongly induced by isoflavone, whereas none of the three flavonoids induced the catabolic pathway of strain LB400. Docking experiments that replaced biphenyl in the biphenyl-bound form of the enzymes with flavone, isoflavone, or flavanone showed that the superior ability of BphAEB356over BphAELB400is principally attributable to the replacement of Phe336 of BphAELB400by Ile334 and of Thr335 of BphAELB400by Gly333 of BphAEB356. However, biochemical and structural comparison of BphAEB356with BphAEp4, a mutant of BphAELB400which was obtained in a previous work by the double substitution Phe336Met Thr335Ala of BphAELB400, provided evidence that other residues or structural features of BphAEB356whose precise identification the docking experiment did not allow are also responsible for the superior catalytic abilities of BphAEB356. Together, these data provide supporting evidence that the biphenyl catabolic pathways have evolved divergently among proteobacteria, where some of them may serve ecological functions related to the metabolism of plant secondary metabolites in soil.

Thiamine pyrophosphate (TPP), a biologically active form of thiamine (vitamin B 1 ), is an essential cofactor in all living systems. Microorganisms either synthesize TPP via de novo biosynthesis pathways or uptake exogenous thiamine from the environment via specific transporters. The oral spirochete Treponema denticola is an important pathogen that is associated with human periodontal diseases. It lacks a de novo TPP biosynthesis pathway and needs exogenous TPP for growth, suggesting that it may obtain exogenous TPP via a thiamine transporter. In this study, we identified a gene cluster that encodes a TPP ABC transporter which consists of a TPP-binding protein (TDE0143), a transmembrane permease (TDE0144), and a cytosolic ATPase (TDE0145). Transcriptional and translational analyses showed that the genes encoding these three proteins are cotranscribed and form an operon (tbpABC Td ) that is initiated by a 70 -like promoter. The expression level of this operon is negatively regulated by exogenous TPP and is mediated by a TPP-sensing riboswitch (Td thi-box ). Genetic and biochemical studies revealed that the TDE0143 deletion mutant (T. denticola ⌬tbpA) had a decreased ability to transport exogenous TPP, and the mutant failed to grow when exogenous TPP was insufficient. These results taken together indicate that the tbpABC Td operon encodes an ABC transporter that is required for the uptake of exogenous TPP and that the expression of this operon is regulated by a TPP-binding riboswitch via a feedback inhibition mechanism.

Summary In the model organism Escherichia coli, the coupling protein CheW, which bridges the chemoreceptors and histidine kinase CheA, is essential for chemotaxis. Unlike the situation in E. coli, Borrelia burgdorferi, the causative agent of Lyme disease, has three cheW homologues (cheW1, cheW2 and cheW3). Here, a comprehensive approach is utilized to investigate the roles of the three cheWs in chemotaxis of B. burgdorferi. First, genetic studies indicated that both the cheW1 and cheW3 genes are essential for chemotaxis, as the mutants had altered swimming behaviours and were non‐chemotactic. Second, immunofluorescence and cryo‐electron tomography studies suggested that both CheW1 and CheW3 are involved in the assembly of chemoreceptor arrays at the cell poles. In contrast to cheW1 and cheW3, cheW2 is dispensable for chemotaxis and assembly of the chemoreceptor arrays. Finally, immunoprecipitation studies demonstrated that the three CheWs interact with different CheAs: CheW1 and CheW3 interact with CheA2 whereas CheW2 binds to CheA1. Collectively, our results indicate that CheW1 and CheW3 are incorporated into one chemosensory pathway that is essential for B. burgdorferi chemotaxis. Although many bacteria have more than one homologue of CheW, to our knowledge, this report provides the first experimental evidence that two CheW proteins coexist in one chemosensory pathway and that both are essential for chemotaxis.

ABSTRACT:Molecular dynamics (MD) simulations of 7-ethoxy-and 7-methoxyresorufin bound in the active site of cytochrome P450 (P450) 1A2 wild-type and various mutants were used to predict changes in substrate specificity of the mutants. A total of 26 multiple mutants representing all possible combinations of five key amino acid residues, which are different between P450 1A1 and 1A2, were examined. The resorufin substrates were docked in the active site of each enzyme in the productive binding orientation, and MD simulations were performed on the enzyme-substrate complex. Ensembles collected from MD trajectories were then scored on the basis of geometric parameters relating substrate position with respect to the activated oxoheme cofactor. The results showed a high correlation between the previous experimental data on P450 1A2 wild-type and single mutants with respect to the ratio between 7-ethoxyresorufin-O-deethylase (EROD) and 7-methoxyresorufin-O-demethylase (MROD) activities and the equivalent in silico "E/M scores" (the ratio of hits obtained with 7-ethoxyresorufin to those obtained with 7-methoxyresorufin). Moreover, this correlation served to establish linear regression models used to evaluate E/M scores of multiple P450 1A2 mutants. Seven mutants, all of them incorporating the L382V substitution, were predicted to shift specificity to that of P450 1A1. The predictions were then verified experimentally. The appropriate P450 1A2 multiple mutants were constructed by site-directed mutagenesis, expressed in Escherichia coli, and assayed for EROD and MROD activities. Of six mutants, five demonstrated an increased EROD/MROD ratio, confirming modeling predictions.Cytochromes P450 constitute a large family of heme-thiolate monooxygenase enzymes widely found in nature (Nelson et al., 1996). These enzymes are capable of catalyzing the oxidation of a wide variety of both xenobiotic and endogenous compounds. However, even closely related isoforms may exhibit different catalytic activities.Human P450 1A1 and 1A2, the two major isoforms in the P450 1A subfamily, share 72% amino acid sequence identity but display different substrate specificities. P450 1A1 prefers to metabolize benzo- [a]pyrene and other polycyclic aromatic hydrocarbons, whereas P450 1A2 favors the oxidation of heterocyclic and aromatic amines (Kawajiri and Hayashi, 1996;Guengerich, 2005). Likewise, they also exhibit different substrate specificities with resorufin substrates such as 7-ethoxyresorufin and 7-methoxyresorufin (Nerurkar et al., 1993;Burke et al., 1994). Therefore, the P450 1A1/1A2 system provides a good model for exploring the basis for functional differences between individual P450 enzymes.The experimentally determined structure of a protein can provide valuable insight into its function. Homology modeling is an alternative method for obtaining the structure when the crystal structure is not available (Szklarz et al., 2000). Molecular dynamics (MD) simulations on the enzyme-substrate complex could provide information on whether the substrat...

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