Evidence that TP_0144 of Treponema pallidum is a thiamine-binding protein.

Thiamine pyrophosphate (TPP), the biologically active form of thiamine (also known as vitamin B1), is an essential cofactor for several important enzymes involved in carbohydrate metabolism, and therefore, it is required for all living organisms. We recently found that a thiamine-binding protein (TDE_0143) is essential for the survival of Treponema denticola, an important bacterial pathogen that is associated with human periodontitis. In this report, we provide experimental evidence showing that TP_0144, a homolog of TDE_0143 from the syphilis spirochete Treponema pallidum, is a thiamine-binding protein that has biochemical features and functions that are similar to those of TDE_0143. First, structural modeling analysis reveal that both TDE_0143 and TP_0144 contain a conserved TPP-binding site and share similar structures to the thiamine-binding protein of Escherichia coli. Second, biochemical analysis shows that these two proteins bind to TPP with similar dissociation constant (Kd) values (TDE_0143, Kd of 36.50 nM; TP_0144, Kd of 32.62 nM). Finally, heterologous expression of TP_0144 in a ΔTDE_0143 strain, a previously constructed TDE_0143 mutant of T. denticola, fully restores its growth and TPP uptake when exogenous thiamine is limited. Collectively, these results indicate that TP_0144 is a thiamine-binding protein that is indispensable for T. pallidum to acquire exogenous thiamine, a key nutrient for bacterial survival. In addition, the studies shown in this report further underscore the feasibility of using T. denticola as a platform to study the biology and pathogenicity of T. pallidum and probably other uncultivable treponemal species as well.

Exploring structurally conservative blocks as universal templates for modeling eukaryotic cytochrome P450s.

BACKGROUND: An increasing number of crystal structures for eukaryotic P450s have been published, which provided the chance to explore more structural features and construct some knowledge-based methods to facilitate modeling. METHODS: The crystal structures of 14 cytochrome P450s (CYP450) were selected to extract generic spatial anchors typical for three-dimensional (3D) structures of eukaryotic P450s. Multiple sequence alignment and structural superimposition were applied to recognize evolutionarily conserved regions. RESULTS: Regions containing uninterrupted helical components were identified as structurally conservative blocks (SCBs). The reliability and robustness of the SCBs were further evaluated by sequence entropy and structural deviation. Finally, these SCBs were applied and tested directly in constructing the homology model of the P450 1B1. CONCLUSIONS: SCBs could potentially be applied as universal template to standardize the homology modeling procedure and help predict drug metabolism preferences for eukaryotic P450s.

Preferred binding orientations of phenacetin in CYP1A1 and CYP1A2 are associated with isoform-selective metabolism.

Human cytochromes P450 1A1 and 1A2 play important roles in drug metabolism and chemical carcinogenesis. Although these two enzymes share high sequence identity, they display different substrate specificities and inhibitor susceptibilities. In the present studies, we investigated the structural basis for these differences with phenacetin as a probe using a number of complementary approaches, such as enzyme kinetics, stoichiometric assays, NMR, and molecular modeling. Kinetic and stoichiometric analyses revealed that substrate specificity (k(cat)/K(m)) of CYP1A2 was approximately 18-fold greater than that of CYP1A1, as expected. Moreover, despite higher H2O2 production, the coupling efficiency of reducing equivalents to acetaminophen formation in CYP1A2 was tighter than that in CYP1A1. CYP1A1, in contrast to CYP1A2, displayed much higher uncoupling, producing more water. The subsequent NMR longitudinal (T1) relaxation studies with the substrate phenacetin and its product acetaminophen showed that both compounds displayed similar binding orientations within the active site of CYP1A1 and CYP1A2. However, the distance between the OCH2 protons of the ethoxy group (site of phenacetin O-deethylation) and the heme iron was 1.5 Å shorter in CYP1A2 than in CYP1A1. The NMR findings are thus consistent with our kinetic and stoichiometric results, providing a likely molecular basis for more efficient metabolism of phenacetin by CYP1A2.

Two CheW coupling proteins are essential in a chemosensory pathway of Borrelia burgdorferi.

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 (cheW(1) , cheW(2) and cheW(3) ). 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 cheW(1) and cheW(3) 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 CheW(1) and CheW(3) are involved in the assembly of chemoreceptor arrays at the cell poles. In contrast to cheW(1) and cheW(3) , cheW(2) is dispensable for chemotaxis and assembly of the chemoreceptor arrays. Finally, immunoprecipitation studies demonstrated that the three CheWs interact with different CheAs: CheW(1) and CheW(3) interact with CheA(2) whereas CheW(2) binds to CheA(1) . Collectively, our results indicate that CheW(1) and CheW(3) 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.

Remarkable ability of Pandoraea pnomenusa B356 biphenyl dioxygenase to metabolize simple flavonoids.

Many 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 BphAE(B356), the biphenyl/chlorobiphenyl dioxygenase from Pandoraea pnomenusa B356, is significantly better fitted to metabolize flavone, isoflavone, and flavanone than BphAE(LB400) from Burkholderia xenovorans LB400. Unlike those of BphAE(LB400), the kinetic parameters of BphAE(B356) toward 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 BphAE(B356) over BphAE(LB400) is principally attributable to the replacement of Phe336 of BphAE(LB400) by Ile334 and of Thr335 of BphAE(LB400) by Gly333 of BphAE(B356). However, biochemical and structural comparison of BphAE(B356) with BphAE(p4), a mutant of BphAE(LB400) which was obtained in a previous work by the double substitution Phe336Met Thr335Ala of BphAE(LB400), provided evidence that other residues or structural features of BphAE(B356) whose precise identification the docking experiment did not allow are also responsible for the superior catalytic abilities of BphAE(B356). 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.

Insight into the metabolism of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) by biphenyl dioxygenases.

In this work we have investigated the ability of the biphenyl dioxygenase of Burkholderia xenovorans LB400 (BphAE(LB400)) and of Pandoraea pnomenusa B356 (BphAE(B356)) to metabolize DDT. Data show BphAE(LB400) is unable to metabolize this substrate but BphAE(B356) metabolizes DDT to produce two stereoisomers. Structural analysis of DDT-docked BphAE(LB400) and BphAE(B356) identified residue Phe336 of BphAE(LB400) as critical to prevent productive binding of DDT to BphAE(LB400). Furthermore, the fact that residue Gly319 of BphAE(B356) is less constrained than Gly321 of BphAE(LB400) most likely contributes to the ability of BphAE(B356) to bind DDT productively. This was confirmed by examining the ability of BphAE chimeras obtained by shuffling bphA genes from strain B356 and LB400. Chimeras where residues Thr335 (which modulates the constraints on Gly321) and Phe336 (which contacts the substrate) of BphAE(LB400) were replaced by Gly and Ile respectively were able to metabolize DDT. However their stereospecificities varied depending on the presence of other segments or residues from BphAE(B356). Structural analysis suggests that either one or both of residue 267 and a segments comprised of residue 247-260 are likely involved in stereospecificity.

The riboswitch regulates a thiamine pyrophosphate ABC transporter of the oral spirochete Treponema denticola.

Thiamine pyrophosphate (TPP), a biologically active form of thiamine (vitamin B1), 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 σ7°-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 ΔtbpA) 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.

Regulation of MMP-2 expression and activity by ß-1,3-N-acetylglucosaminyltransferase-8 in AGS gastric cancer cells.

ß-1,3-N-acetylglucosaminyltransferase-8(ß3Gn-T8) catalyzes the transfer of GlcNAc to the non-reducing terminus of the Galß1-4GlcNAc of tetraantennary N-glycan in vitro. It has been reported to be involved in malignant tumors, but a comprehensive understanding of how the glycolsyltransferase correlates with the invasive potential of human gastric cancer is not currently available. Therefore, we investigated the ability and possible mechanism involved with ß3Gn-T8 in modulating matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) in AGS gastric cancer cells. Here, we found out that siRNA-mediated suppression of the ß3Gn-T8 could directly reduce the MMP-2 expression and activity as observed in RT-PCR, western blot and gelatin zymography analysis. Meanwhile, TIMP-2 expression had been increased. Cell invasion assay using matrigel matrix-coated transwell inserts showed that the invasive property was greatly suppressed in ß3Gn-T8 siRNA transfected cells. Furthermore, cells overexpressing ß3Gn-T8 gene (when transfected with pEGFP-C1 plasmid) also expressed MMP-2 gene, but TIMP-2 expression had been inhibited. The invasive ability of these cells was also enhanced. Protein-protein interaction analysis using STRING database showed that ß3Gn-T8 and MMP-2 may have related signal pathway. In summary, our results reveal a new mechanism by which ß3Gn-T8 can regulate MMP-2 and TIMP-2. We suggest that ß3Gn-T8 can be used as a novel therapeutic target for human gastric treatment.

Expression and functional analysis of CYP2D6.24, CYP2D6.26, CYP2D6.27, and CYP2D7 isozymes.

The objectives of this study were to compare the drug-metabolizing activity of human CYP2D6.24 (I297L), CYP2D6.26 (I369T), and CYP2D6.27 (E410K) allelic isoforms with wild-type CYP2D6.1 and to express the CYP2D7 protein derived from an indel polymorphism (CYP2D7 138delT) and investigate its possible codeine O-demethylase activity. Successful creation of individual cDNAs corresponding to CYP2D6*24 (2853 A>C), CYP2D6*26 (3277 T>C), and CYP2D6*27 (3853 G>A) allelic variants and CYP2D7 was achieved via molecular cloning. The corresponding proteins, CYP2D6.24, CYP2D6.26, CYP2D6.27, and CYP2D7, were expressed in insect cells by using a baculovirus-mediated expression system. All CYP2D proteins showed the empirical carbon monoxide difference spectra. We were surprised to find that the CYP2D7 protein was detected mainly in mitochondrial fractions, whereas all CYP2D6 allelic isoforms were present in the microsomal fraction. Furthermore, CYP2D7 did not produce any morphine from codeine. In contrast, CYP2D6.24, CYP2D6.26, and CYP2D6.27 allelic isoforms all showed active drug-metabolizing activities toward both codeine and dextromethorphan O-demethylation. Whereas CYP2D6.24 exhibited the highest intrinsic clearance in dextromethorphan O-demethylation (approximately 6-fold higher than that by CYP2D6.1), it had the lowest enzyme efficiency in codeine O-demethylation (approximately 50% lower than that by CYP2D6.1). Overall, the enzymatic consequences of CYP2D6 allelic isozymes are substrate dependent. These data would help preclinical and clinical assessments of the metabolic elimination of drugs that are mediated by human CYP2D enzyme.

Application of molecular modeling for prediction of substrate specificity in cytochrome P450 1A2 mutants.

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.