http://www.biomedcentral.com/bmcbiochem/ The latest research articles published by BMC Biochemistry 2009-06-24T00:00:00Z This is an RSS newsfeed from BioMed Central It is intended to be used with an RSS reader. For more information about RSS newsfeeds from BioMed Central, visit http://www.biomedcentral.com/info/about/rss/ Background: Malate synthase catalyzes the second step of the glyoxylate bypass, the condensation of acetyl coenzyme A and glyoxylate to form malate and coenzyme A (CoA). In several microorganisms, the glyoxylate bypass is of general importance to microbial pathogenesis. The predicted malate synthase G of Pseudomonas aeruginosa has also been implicated in virulence of this opportunistic pathogen. Results: Here, we report the verification of the malate synthase activity of this predicted protein and its recombinant production in E. coli, purification and biochemical characterization. The malate synthase G of P. aeruginosa PAO1 has a temperature and pH optimum of 37.5°C and 8.5, respectively. Although displaying normal thermal stability, the enzyme was stable up to incubation at pH 11. The following kinetic parameters of P. aeruginosa PAO1 malate synthase G were obtained: Km glyoxylate (70 μM), Km acetyl CoA (12 μM) and Vmax (16.5 μmol/minutes/mg enzyme). In addition, deletion of the corresponding gene showed that it is a prerequisite for growth on acetate as sole carbon source. Conclusion: The implication of the glyoxylate bypass in the pathology of various microorganisms makes malate synthase G an attractive new target for antibacterial therapy. The purification procedure and biochemical characterization assist in the development of antibacterial components directed against this target in P. aeruginosa. http://www.biomedcentral.com/1471-2091/10/20 Bart Roucourt Nikki Minnebo Patrick Augustijns Kirsten Hertveldt Guido Volckaert Rob Lavigne BMC Biochemistry 2009, 10:20 2009-06-24T00:00:00Z doi:10.1186/1471-2091-10-20 BMC Biochemistry 1471-2091 10 20 2009-06-24T00:00:00Z XML Background: Escherichia coli MutY (EcMutY) reduces mutagenesis by removing adenines paired with guanines or 7,8-dihydro-8-oxo-guanines (8-oxoG). V45 and Q182 of EcMutY are considered the key determinants of adenine specificity. Both residues are spatially close to each other in the active site and are conserved in MutY family proteins but not to Methanobacterium thermoautotrophicum Mig.MthI T/G mismatch DNA glycosylase (A50 and L187 at the corresponding respective positions). Results: Targeted mutagenesis study was performed to determine the substrate specificities of V45A, Q182L, and V45A/Q182L double mutant. All three mutants had significantly lower binding and glycosylase activities for A/G and A/8-oxoG mismatches than the wild-type enzyme. The double mutant exhibited an additive reduction in binding to both the A/G and A/GO as for the single mutants. These mutants were also tested for binding and glycosylase activities with T/G- or T/8-oxoG-containing DNA. Both V45A and Q182L mutants had increased affinities towards T/G, however, they did not exhibit any T/G or T/8-oxoG glycosylase activity. Surprisingly, the V45A/Q182L double mutant had similar binding affinities to T/G and T/8-oxoG as the wild-type EcMutY. V45A, Q182L, and V45A/Q182L EcMutY mutants could not reduce the G:C to T:A mutation frequency of a mutY mutant. Expression of the V45A mutant protein caused a dominant negative phenotype with an increased G:C to A:T mutation frequency. Conclusion: The substrate specificities are altered in V45A, Q182L, and V45A/Q182L EcMutY mutants. V45A and Q182L mutants had reduced binding and glycosylase activities for A/G and A/8-oxoG mismatches and increased affinities towards T/G mismatch. However, in contrast to a previous report that Mig.MthI thymine DNA glycosylase can be converted to a MutY-like adenine glycosylase by replacing two residues (A50V and L187Q), both V45A and Q182L EcMutY mutants did not exhibit any T/G or T/8-oxoG glycosylase activity. The dominant negative phenotype of V45A EcMutY mutant protein is probably caused by its increased binding affinity to T/G mismatch and thus inhibiting other repair pathways. http://www.biomedcentral.com/1471-2091/10/19 Po-Wen Chang Amrita Madabushi A-Lien Lu BMC Biochemistry 2009, 10:19 2009-06-12T00:00:00Z doi:10.1186/1471-2091-10-19 BMC Biochemistry 1471-2091 10 19 2009-06-12T00:00:00Z PDF Background: Mycobacterium tuberculosis DNA topoisomerase I is an attractive target for discovery of novel TB drugs that act by enhancing the accumulation of the topoisomerase-DNA cleavage product. It shares a common transesterification domain with other type IA DNA topoisomerases. There is, however, no homology between the C-terminal DNA binding domains of Escherichia coli and M. tuberculosis DNA topoisomerase I proteins. Results: A new protocol for expression and purification of recombinant M. tuberculosis DNA topoisomerase I (MtTOP) has been developed to produce enzyme of much higher specific activity than previously characterized recombinant enzyme. MtTOP was found to be less efficient than E. coli DNA topoisomerase I (EcTOP) in removal of remaining negative supercoils from partially relaxed DNA. DNA cleavage by MtTOP was characterized for the first time. Comparison of DNA cleavage site selectivity with EcTOP showed differences in cleavage site preferences, but the preferred sites of both enzymes have a C nucleotide in the -4 position. Conclusion: Recombinant M. tuberculosis DNA topoisomerase I can be expressed as a soluble protein and purified in high yield from E. coli host with a new protocol. Analysis of DNA cleavage with M. tuberculosis DNA substrate showed that the preferred DNA cleavage sites have a C nucleotide in the -4 position. http://www.biomedcentral.com/1471-2091/10/18 Thirunavukkarasu Annamalai Neil Dani Bokun Cheng Yuk-Ching Tse-Dinh BMC Biochemistry 2009, 10:18 2009-06-11T00:00:00Z doi:10.1186/1471-2091-10-18 BMC Biochemistry 1471-2091 10 18 2009-06-11T00:00:00Z XML Background: Bacteriocin production in the lactic acid bacterium Lactobacillus plantarum C11 is regulated through a quorum sensing based pathway involving two highly homologous response regulators (59% identity and 76% similarity), PlnC as a transcriptional activator and PlnD as a repressor. Previous in vitro studies have shown that both regulators bind, as homodimers, to the same DNA regulatory repeats to exert their regulatory functions. As the genes for these two proteins are located on the same auto-regulatory operon, hence being co-expressed upon gene activation, it is plausible that their opposite functions must somehow be differentially regulated, either in terms of timing and/or binding kinetics, so that their activities do not impair each other in an uncontrolled manner. To understand the nature behind this potential differentiation, we have studied the binding kinetics of the two regulators on five target promoters (PplnA, PplnM, PplnJ, PplnE and PplnG) from the bacteriocin regulon of L. plantarum C11. Results: By using surface plasmon resonance spectroscopy we obtained parameters such as association rates, dissociation rates and dissociation constants, showing that the two regulators indeed differ greatly from each other in terms of cooperative binding and binding strength to the different promoters. For instance, cooperativity is very strong for PlnC binding to the promoter of the regulatory operon (PplnA), but not to the promoter of the transport operon (PplnG), while the opposite is seen for PlnD binding to these two promoters. The estimated affinity constants indicate that PlnC can bind to PplnA to activate transcription of the key regulatory operon plnABCD without much interference from PlnD, and that the repressive function of PlnD might act through a different mechanism than repression of the regulatory operon. Conclusion: We have characterised the DNA binding kinetics of the two regulators PlnC and PlnD from the bacteriocin locus in L. plantarum C11. Our data show that PlnC and PlnD, despite their strong homology to each other, differ greatly from each other in terms of binding affinity and cooperativity to the different promoters of the pln regulon. http://www.biomedcentral.com/1471-2091/10/17 Daniel Straume Rune Johansen Magnar Bjoras Ingolf Nes Dzung Diep BMC Biochemistry 2009, 10:17 2009-06-11T00:00:00Z doi:10.1186/1471-2091-10-17 BMC Biochemistry 1471-2091 10 17 2009-06-11T00:00:00Z PDF Background: The re-replication inhibitor Geminin binds to several transcription factors including homeodomain proteins, and to members of the polycomb and the SWI/SNF complexes. Results: Here we describe the TATA-binding protein-like factor-interacting protein (TIPT) isoform 2, as a strong binding partner of Geminin. TIPT2 is widely expressed in mouse embryonic and adult tissues, residing both in cyto- and nucleoplasma, and enriched in the nucleolus. Like Geminin, also TIPT2 interacts with several polycomb factors, with the general transcription factor TBP (TATA box binding protein), and with the related protein TBPL1 (TRF2). TIPT2 synergizes with geminin and TBP in the activation of TATA box-containing promoters, and with TBPL1 and geminin in the activation of the TATA-less NF1 promoter. Geminin and TIPT2 were detected in the chromatin near TBP/TBPL1 binding sites. Conclusion: Together, our study introduces a novel transcriptional regulator and its function in cooperation with chromatin associated factors and the basal transcription machinery. http://www.biomedcentral.com/1471-2091/10/16 Mara Pitulescu Martin Teichmann Lingfei Luo Michael Kessel BMC Biochemistry 2009, 10:16 2009-06-10T00:00:00Z doi:10.1186/1471-2091-10-16 BMC Biochemistry 1471-2091 10 16 2009-06-10T00:00:00Z XML Background: Protein acetylation is among the most common protein modifications. The two major types are post-translational Nε-lysine acetylation catalyzed by KATs (Lysine acetyltransferases, previously named HATs (histone acetyltransferases) and co-translational Nα-terminal acetylation catalyzed by NATs (N-terminal acetyltransferases). The major NAT complex in yeast, NatA, is composed of the catalytic subunit Naa10p (N alpha acetyltransferase 10 protein) (Ard1p) and the auxiliary subunit Naa15p (Nat1p). The NatA complex potentially acetylates Ser-, Ala-, Thr-, Gly-, Val- and Cys- N-termini after Met-cleavage. In humans, the homologues hNaa15p (hNat1) and hNaa10p (hArd1) were demonstrated to form a stable ribosome associated NAT complex acetylating NatA type N-termini in vitro and in vivo. Results: We here describe a novel human protein, hNaa16p (hNat2), with 70% sequence identity to hNaa15p (hNat1). The gene encoding hNaa16p originates from an early vertebrate duplication event from the common ancestor of hNAA15 and hNAA16. Immunoprecipitation coupled to mass spectrometry identified both endogenous hNaa15p and hNaa16p as distinct interaction partners of hNaa10p in HEK293 cells, thus demonstrating the presence of both hNaa15p-hNaa10p and hNaa16p-hNaa10p complexes. The hNaa16p-hNaa10p complex acetylates NatA type N-termini in vitro. hNaa16p is ribosome associated, supporting its potential role in cotranslational Nα-terminal acetylation. hNAA16 is expressed in a variety of human cell lines, but is generally less abundant as compared to hNAA15. Specific knockdown of hNAA16 induces cell death, suggesting an essential role for hNaa16p in human cells. Conclusion: At least two distinct NatA protein Nα-terminal acetyltransferases coexist in human cells potentially creating a more complex and flexible system for Nα-terminal acetylation as compared to lower eukaryotes. http://www.biomedcentral.com/1471-2091/10/15 Thomas Arnesen Darina Gromyko Diane Kagabo Matthew Betts Kristian Starheim Jan Erik Varhaug Dave Anderson Johan Lillehaug BMC Biochemistry 2009, 10:15 2009-05-29T00:00:00Z doi:10.1186/1471-2091-10-15 BMC Biochemistry 1471-2091 10 15 2009-05-29T00:00:00Z XML Background: N-arachidonoyl glycine (NAGly) is an endogenous signaling lipid with a wide variety of biological activity whose biosynthesis is poorly understood. Two primary biosynthetic pathways have been proposed. One suggests that NAGly is formed via an enzymatically regulated conjugation of arachidonic acid (AA) and glycine. The other suggests that NAGly is an oxidative metabolite of the endogenous cannabinoid, anandamide (AEA), through an alcohol dehydrogenase. Here using both in vitro and in vivo assays measuring metabolites with LC/MS/MS we test the hypothesis that both pathways are present in mammalian cells. Results: The metabolic products of deuterium-labeled AEA, D4AEA (deuterium on ethanolamine), indicated that NAGly is formed by the oxidation of the ethanolamine creating a D2NAGly product in both RAW 264.7 and C6 glioma cells. Significantly, D4AEA produced a D0NAGly product only in C6 glioma cells suggesting that the hydrolysis of AEA yielded AA that was used preferentially in a conjugation reaction. Addition of the fatty acid amide (FAAH) inhibitor URB 597 blocked the production of D0NAGly in these cells. Incubation with D8AA in C6 glioma cells likewise produced D8NAGly; however, with significantly less efficacy leading to the hypothesis that FAAH-initiated AEA-released AA conjugation with glycine predominates in these cells. Furthermore, the levels of AEA in the brain were significantly increased, whereas those of NAGly were significantly decreased after systemic injection of URB 597 in rats and in FAAH KO mice further supporting a role for FAAH in endogenous NAGly biosynthesis. Incubations of NAGly and recombinant FAAH demonstrated that NAGly is a significantly less efficacious substrate for FAAH with only ~50% hydrolysis at 30 minutes compared to 100% hydrolysis of AEA. Co-incubations of AEA and glycine with recombinant FAAH did not, however, produce NAGly. Conclusion: These data support the hypothesis that the signaling lipid NAGly is a metabolic product of AEA by both oxidative metabolism of the AEA ethanolamine moiety and through the conjugation of glycine to AA that is released during AEA hydrolysis by FAAH. http://www.biomedcentral.com/1471-2091/10/14 Heather Bradshaw Neta Rimmerman Sherry Shu-Jung Hu Valery Benton Jordyn Stuart Kim Masuda Benjamin Cravatt David O'Dell J Michael Walker BMC Biochemistry 2009, 10:14 2009-05-21T00:00:00Z doi:10.1186/1471-2091-10-14 BMC Biochemistry 1471-2091 10 14 2009-05-21T00:00:00Z XML Background: TAFI is a plasma protein assumed to be an important link between coagulation and fibrinolysis. The three-dimensional crystal structures of authentic mature bovine TAFI (TAFIa) in complex with tick carboxypeptidase inhibitor, authentic full lenght bovine plasma thrombin-activatable fibrinolysis inhibitor (TAFI), and recombinant human TAFI have recently been solved. In light of these recent advances, we have characterized authentic bovine TAFI biochemically and compared it to human TAFI. Results: The four N-linked glycosylation sequons within the activation peptide were all occupied in bovine TAFI, similar to human TAFI, while the sequon located within the enzyme moiety of the bovine protein was non-glycosylated. The enzymatic stability and the kinetic constants of TAFIa differed somewhat between the two proteins, as did the isoelectric point of TAFI, but not TAFIa. Equivalent to human TAFI, bovine TAFI was a substrate for transglutaminases and could be proteolytically cleaved by trypsin or thrombin/solulin complex, although small differences in the fragmentation patterns were observed. Furthermore, bovine TAFI exhibited intrinsic activity and TAFIa attenuated tPA-mediated fibrinolysis similar to the human protein. Conclusion: The findings presented here suggest that the properties of these two orthologous proteins are similar and that conclusions reached using the bovine TAFI may be extrapolated to the human protein. http://www.biomedcentral.com/1471-2091/10/13 Zuzana Valnickova Morten Thaysen-Andersen Peter Hojrup Trine Christensen Kristian Sanggaard Torsten Kristensen Jan Enghild BMC Biochemistry 2009, 10:13 2009-05-05T00:00:00Z doi:10.1186/1471-2091-10-13 BMC Biochemistry 1471-2091 10 13 2009-05-05T00:00:00Z XML Background: The ALG2-interacting protein X (ALIX)/AIP1 is an adaptor protein with multiple functions in intracellular protein trafficking that plays a central role in the biogenesis of enveloped viruses. The ubiquitin E3-ligase POSH (plenty of SH3) augments HIV-1 egress by facilitating the transport of Gag to the cell membrane. Recently, it was reported, that POSH interacts with ALIX and thereby enhances ALIX mediated phenotypes in Drosophila. Results: In this study we identified ALIX as a POSH ubiquitination substrate in human cells: POSH induces the ubiquitination of ALIX that is modified on several lysine residues in vivo and in vitro. This ubiquitination does not destabilize ALIX, suggesting a regulatory function. As it is well established that ALIX rescues virus release of L-domain mutant HIV-1, HIV-1ΔPTAP, we demonstrated that wild type POSH, but not an ubiquitination inactive RING finger mutant (POSHV14A), substantially enhances ALIX-mediated release of infectious virions derived from HIV-1ΔPTAP L-domain mutant (YPXnL-dependent HIV-1). In further agreement with the idea of a cooperative function of POSH and ALIX, mutating the YPXnL-ALIX binding site in Gag completely abrogated augmentation of virus release by overexpression of POSH. However, the effect of the POSH-mediated ubiquitination appears to be auxiliary, but not necessary, as silencing of POSH by RNAi does not disturb ALIX-augmentation of virus release. Conclusion: Thus, the cumulative results identified ALIX as an ubiquitination substrate of POSH and indicate that POSH and ALIX cooperate to facilitate efficient virus release. However, while ALIX is obligatory for the release of YPXnL-dependent HIV-1, POSH, albeit rate-limiting, may be functionally interchangeable. http://www.biomedcentral.com/1471-2091/10/12 Joerg Voetteler Elena Iavnilovitch Orit Fingrut Vivian Shemesh Daniel Taglicht Omri Erez Stefan Sorgel Torsten Walther Norbert Bannert Ulrich Schubert Yuval Reiss BMC Biochemistry 2009, 10:12 2009-04-24T00:00:00Z doi:10.1186/1471-2091-10-12 BMC Biochemistry 1471-2091 10 12 2009-04-24T00:00:00Z XML Background: Human S100A12 is a member of the S100 family of EF-hand calcium-modulated proteins that are associated with many diseases including cancer, chronic inflammation and neurological disorders. S100A12 is an important factor in host/parasite defenses and in the inflammatory response. Like several other S100 proteins, it binds zinc and copper in addition to calcium. Mechanisms of zinc regulation have been proposed for a number of S100 proteins e.g. S100B, S100A2, S100A7, S100A8/9. The interaction of S100 proteins with their targets is strongly dependent on cellular microenvironment. Results: The aim of the study was to explore the factors that influence S100A12 oligomerization and target interaction. A comprehensive series of biochemical and biophysical experiments indicated that changes in the concentration of calcium and zinc led to changes in the oligomeric state of S100A12. Surface plasmon resonance confirmed that the presence of both calcium and zinc is essential for the interaction of S100A12 with one of its extracellular targets, RAGE – the Receptor for Advanced Glycation End products. By using a single-molecule approach we have shown that the presence of zinc in tissue culture medium favors both the oligomerization of exogenous S100A12 protein and its interaction with targets on the cell surface. Conclusion: We have shown that oligomerization and target recognition by S100A12 is regulated by both zinc and calcium. Our present work highlighted the potential role of calcium-binding S100 proteins in zinc metabolism and, in particular, the role of S100A12 in the cross talk between zinc and calcium in cell signaling. http://www.biomedcentral.com/1471-2091/10/11 Olga Moroz Will Burkitt Helmut Wittkowski Wei He Anatoli Ianoul Vera Novitskaya Jingjing Xie Oxana Polyakova Igor Lednev Alexander Shekhtman Peter Derrick Per Bjoerk Dirk Foell Igor Bronstein BMC Biochemistry 2009, 10:11 2009-04-23T00:00:00Z doi:10.1186/1471-2091-10-11 BMC Biochemistry 1471-2091 10 11 2009-04-23T00:00:00Z XML