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Biochemical Journal

Dipanwita Bhattacharya, Kanchan Sinha, Dulal Panda
The role of FtsZ-associated proteins in the regulation of the assembly dynamics of Mycobacterium smegmatis FtsZ is not clear.  In this work, we examined the effect of Mycobacterium smegmatis SepF on the assembly and stability of Mycobacterium smegmatis FtsZ polymers. We discovered a single dominant point mutation in SepF (G51D or G51R) that renders the protein inactive. SepF promoted the polymerization of FtsZ, induced the bundling of FtsZ filaments, stabilized FtsZ filaments and reduced the GTPase activity of FtsZ...
July 13, 2018: Biochemical Journal
Dirk Maurer, Bernhard Lohkamp, Michael Krumpel, Mikael Widersten, Doreen Dobritzsch
β-Ureidopropionase catalyzes the third step of the reductive pyrimidine catabolic pathway responsible for breakdown of uracil, thymine and pyrimidine-based antimetabolites such as 5-fluorouracil. Nitrilase-like β-ureidopropionases use a tetrad of conserved residues (Cys233, Lys196, Glu119 and Glu207) for catalysis and occur in a variety of oligomeric states. Positive cooperativity towards the substrate N-carbamoyl-β-alanine and an oligomerization-dependent mechanism of substrate activation and product inhibition have been reported for the enzymes from some species but not others...
July 5, 2018: Biochemical Journal
Olawale G Raimi, Ramón Hurtado Guerrero, Daan Mf van Aalten
N -acetylphosphoglucosamine mutase (AGM1) is a key component of the hexosamine biosynthetic pathway that produces UDP-GlcNAc, an essential precursor for a wide range of glycans in eukaryotes. AGM belongs to the a-D-phosphohexomutase metalloenzyme superfamily and catalyses the interconversion of N -acetylglucosamine-6-phosphate (GlcNAc-6P) to N -acetylglucosamine-1-phosphate (GlcNAc-1P) through N -acetylglucosamine-1,6-bisphosphate (GlcNAc-1,6-bisP) as the catalytic intermediate. Although there is an understanding of the phosphoserine-dependent catalytic mechanism at enzymatic and structural level, the identity of the requisite catalytic base in AGM1/phosphoglucomutases is as yet unknown...
July 2, 2018: Biochemical Journal
Jagadish P Hazra, Nisha Arora, Amin Sagar, Shwetha Srinivasan, Abhishek Chaudhuri, Sabyasachi Rakshit
Mechanical cues often influence the factors affecting the transition-states of catalytic reactions and alter the activation-pathway. However, tracking the real-time dynamics of such activation-pathways is limited. Using single-molecule trapping of reaction-intermediate, we developed a method that enabled us to perform one reaction at one site and simultaneously study the real-time dynamics of the catalytic pathway. Using this, we showed single-molecule calligraphy at nanometer resolution and deciphered the mechanism of sortase A enzymatic reaction that counterintuitively accelerates bacterial-adhesion under shear-tension...
July 2, 2018: Biochemical Journal
R Srikalaivani, Amrita Singh, Avadhesha Surolia, M Vijayan
Biochemical and crystallographic studies on Mycobacterium tuberculosis 3-hydroxyisobutyric acid dehydrogenase ( Mt HIBADH), a member of the 3-hydroxyacid dehydrogenase superfamily, have been carried out. Gel filtration and blue native PAGE of Mt HIBADH show that the enzyme is a dimer. The enzyme preferentially uses NAD+ as the cofactor and is specific to S- hydroxyisobutyric acid (HIBA). It can also use R- HIBA, L- serine and 3- hydroxypropanoic acid (3-HP) as substrates, but with much less efficiency. The pH optimum for activity is around 11...
June 29, 2018: Biochemical Journal
Xuaunkun Zeng, Arzu Uyar, Dexin Sui, Nazanin Donyapour, Dianqing Wu, Alex Dickson, Jian Hu
Signaling molecule phosphatidylinositol 4,5-bisphosphate (PIP2 ) is produced primarily by phosphatidylinositol 4-phosphate 5-kinase (PIP5K). PIP5K is essential for the development of the human neuronal system, which has been exemplified by a recessive genetic disorder, lethal congenital contractural syndrome type 3 (LCCS3), caused by a single aspartate-to-asparagine mutation in the kinase domain of PIP5Kγ. So far, the exact role of this aspartate residue has yet to be elucidated. In this work, we conducted structural, functional and computational studies on a zebrafish PIP5Kα variant with a mutation at the same site...
June 29, 2018: Biochemical Journal
Albert G Remacle, Swathi K Hullugundi, Jennifer Dolkas, Mila Angert, Piotr Cieplak, David Scott, Andrei V Chernov, Veronica I Shubayev, Alex Y Strongin
In demyelinating nervous system disorders, myelin basic protein (MBP), a major component of the myelin sheath, is proteolyzed and its fragments are released in the neural environment. Here, we demonstrated that, in contrast with MBP, the cellular uptake of the cryptic 84-104 epitope (MBP84-104) did not involve the low-density lipoprotein receptor-related protein-1, a scavenger receptor. Our pull-down, mass-spectrometry and molecular modeling studies suggested that, similar with a number of other unfolded and aberrant proteins and peptides, the internalized MBP84-104 was capable of binding to the voltage-dependent anion-selective channel-1 (VDAC-1), a mitochondrial porin...
June 28, 2018: Biochemical Journal
Dominic P Byrne, Yong Li, Krithika Ramakrishnan, Igor L Barsukov, Edwin A Yates, Claire E Eyers, Dulcé Papy-Garcia, Sandrine Chantepie, Vijayakanth Pagadala, Jian Lu, Carrow Wells, David H Drewry, William J Zuercher, Neil G Berry, David G Fernig, Patrick A Eyers
Sulphation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulphate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulphotransferases, including heparan sulphate 2- O -sulphotransferase (HS2ST), which transfers sulphate from the co-factor PAPS (3'-phosphoadenosine 5'-phosphosulphate) to the 2- O  position of a-L-iduronate in the maturing polysaccharide chain...
June 22, 2018: Biochemical Journal
Dominic P Byrne, Yong Li, Pawin Ngamlert, Krithika Ramakrishnan, Claire E Eyers, Carrow Wells, David H Drewry, William J Zuercher, Neil G Berry, David G Fernig, Patrick A Eyers
Protein tyrosine sulphation is a post-translational modification best known for regulating extracellular protein-protein interactions. Tyrosine sulphation is catalysed by two Golgi-resident enzymes termed Tyrosyl Protein Sulpho Transferases (TPSTs) 1 and 2, which transfer sulphate from the co-factor PAPS (3'-phosphoadenosine 5'-phosphosulphate) to a context-dependent tyrosine in a protein substrate. A lack of quantitative tyrosine sulphation assays has hampered the development of chemical biology approaches for the identification of small molecule inhibitors of tyrosine sulphation...
June 22, 2018: Biochemical Journal
José L Neira, María Belén López, Paz Sevilla, Bruno Rizzuti, Ana Cámara-Artigas, Miguel Vidal, Juan L Iovanna
NUPR1 is a protumoral multifunctional intrinsically disordered protein (IDP), which is activated during the acute phases of pancreatitis. It interacts with other IDPs such as prothymosin α, as well as with folded proteins such as the C-terminal region of RING1-B (C-RING1B) of the Polycomb complex; in all those interactions, residues around Ala33 and Thr68 (the "hot-spot" region) of NUPR1 intervene. Its paralogue, NUPR1L, is also expressed in response to DNA-damage, it is p53-regulated, and its expression down-regulates that of the NUPR1 gene...
June 20, 2018: Biochemical Journal
Mohammad Kasom, Samia Gharra, Isra Sadiya, Meirav Avital-Shacham, Mickey Kosloff
Regulators of G protein Signaling (RGS) proteins inactivate Gα subunits, thereby controling G protein-coupled signaling networks. Among all RGS proteins, RGS2 is unique in interacting only with the Gαq and not with the Gαi sub-family. Previous studies suggested that this specificity is determined by the RGS domain, and in particular by three RGS2-specific residues that lead to a unique mode of interaction with Gαq This interaction was further proposed to act through contacts with the Gα GTPase domain. Here, we combined energy calculations and GTPase activity measurements to determine which Gα residues dictate specificity toward RGS2...
June 20, 2018: Biochemical Journal
Freyr Jóhannsson, Steinn Guðmundsson, Giuseppe Paglia, Sveinn Guðmundsson, Bernhard Palsson, Ólafur E Sigurjónsson, Óttar Rolfsson
Platelets deteriorate over time when stored within blood banks through a biological process known as platelet storage lesion (PSL). Here we describe the refinement of biochemical network of platelet metabolism iAT-PLT-636 and its application to describe and investigate changes in metabolism during platelet storage. Changes to extracellular acetate and citrate were measured in buffy coat and apheresis platelet units over 10 days of storage in the platelet additive solution T-Sol. Metabolic network analysis of these data was performed alongside our prior metabolomics data to describe the metabolism of fresh (days 1-3), intermediate (days 4-6), and expired (days 7-10) platelets...
June 18, 2018: Biochemical Journal
Biancamaria Farina, Mattia Sturlese, Fabiola Mascanzoni, Andrea Caporale, Alessandra Monti, Gianluigi Di Sorbo, Roberto Fattorusso, Menotti Ruvo, Nunzianna Doti
The complex formation between the proteins Apoptosis Inducing Factor (AIF) and Cyclophilin A (CypA) following oxidative stress in neuronal cells has been suggested as a main target for reverting ischemia-stroke damage. Recently, a peptide encompassing AIF residues 370-394 has been developed to target the AIF-binding site on CypA, to prevent the association between the two proteins and suppress glutamate-induced cell death in neuronal cells. Using a combined approach based on NMR spectroscopy, synthesis and in vitro testing of all Ala-scan mutants of the peptide and molecular docking/molecular dynamics, we have generated a detailed model of the AIF(370-394)/CypA complex...
June 11, 2018: Biochemical Journal
Pengpeng Li, Dongqing Xu, Tiequn Ma, Daoying Wang, Weidong Li, Jianhua He, Tingting Ran, Weiwu Wang
The type VI secretion system (T6SS) is a versatile and widespread export system found in many Gram-negative bacteria that delivers effector proteins into target cells. The functions of T6SSs are tightly regulated by diverse mechanisms at multiple levels, including posttranslational modification through threonine phosphorylation via the Ser/Thr protein kinase (STPK) PpkA. Here, we identified that PpkA is essential for T6SS secretion in Serratia marcescens since its deletion eliminated the secretion of hemolysin coregulated protein (Hcp), while the periplasmic and transmembrane portion of PpkA was found to be disposable for T6SS secretion...
June 1, 2018: Biochemical Journal
Ingrid J G Burvenich, William Farrugia, Zhanqi Liu, Dahna Makris, Dylan King, Benjamin Gloria, Angelo Perani, Laura C Allan, Andrew M Scott, Paul A Ramsland
Antibody engineering is important for many diagnostic and clinical applications of monoclonal antibodies. We recently reported a series of Fc mutations targeting the neonatal Fc receptor (FcRn) site on a Lewis Y binding IgG1, hu3S193. The hu3S193 variants displayed shortened in vivo half-lives and may have potential for radioimaging or radiotherapy of Lewis Y positive tumors. Here we report Fc crystal structures of wild-type hu3S193, seven FcRn binding site variants, and a variant lacking C1q binding or complement-dependent cytotoxicity (CDC) activity...
May 24, 2018: Biochemical Journal
Min-Kyu Kwak, Han-Bong Ryu, Sung-Hyun Song, Jin-Won Lee, Sa-Ouk Kang
YlaD, a membrane-anchored anti-sigma factor of Bacillus subtilis , contains a HX3 CXXC motif that functions as a redox-sensing domain and belongs to one of the zinc-coordinated anti-sigma factor families. Despite previously showing that the YlaC transcription is controlled by YlaD, experimental evidence of how the YlaC-YlaD interaction is affected by active cysteines and/or metal ions is lacking. Here, we showed that the P yla promoter is autoregulated solely by YlaC. Moreover, reduced YlaD contained zinc and iron, while oxidized YlaD did not...
May 14, 2018: Biochemical Journal
Makoto Kurano, Hitoshi Ikeda, Naoyuki Iso-O, Masumi Hara, Kazuhisa Tsukamoto, Yutaka Yatomi
Apolipoprotein M (apoM) is a carrier and a modulator of sphingosine 1-phosphate (S1P), an important multi-functional bioactive lipid. Since PPARγ is reportedly associated with the function and metabolism of S1P, we investigated the modulation of apoM/S1P homeostasis by PPARγ. First, we investigated the modulation of apoM and S1P homeostasis by the overexpression or knockdown of PPARγ in HepG2 cells and found that both the overexpression and the knockdown of PPARγ decreased apoM expression and S1P synthesis...
April 30, 2018: Biochemical Journal
Abi S Ghifari, Mabel Gill-Hille, Monika W Murcha
The majority of the mitochondrial proteome, required to fulfil its diverse range of functions, is cytosolically synthesised and translocated via specialised machinery. The dedicated translocases, receptors, and associated proteins have been characterised in great detail in yeast over the last several decades, yet many of the mechanisms that regulate these processes in higher eukaryotes are still unknown. In this review, we highlight the current knowledge of mitochondrial protein import in plants. Despite the fact that the mechanisms of mitochondrial protein import have remained conserved across species, many unique features have arisen in plants to encompass the developmental, tissue-specific, and stress-responsive regulation in planta...
July 17, 2018: Biochemical Journal
Jeffrey M Arciola, Nicole A Horenstein
Biosynthesis of the azasugar 1-deoxynojirimycin (DNJ) critically involves a transamination in the first committed step. Here, we identify the azasugar biosynthetic cluster signature in Paenibacillus polymyxa SC2 ( Ppo ), homologous to that reported in Bacillus amyloliquefaciens FZB42 ( Bam ), and report the characterization of the aminotransferase GabT1 (named from Bam ). GabT1 from Ppo exhibits a specific activity of 4.9 nmol/min/mg at 30°C (pH 7.5), a somewhat promiscuous amino donor selectivity, and curvilinear steady-state kinetics that do not reflect the predicted ping-pong behavior typical of aminotransferases...
July 17, 2018: Biochemical Journal
Raimund Nagel, Reuben J Peters
Biosynthesis of the gibberellin A (GA) plant hormones evolved independently in plant-associated fungi and bacteria. While the relevant enzymes have distinct evolutionary origins, the pathways proceed via highly similar reactions. One particularly complex transformation involves combined demethylation and γ-lactone ring formation, catalyzed in bacteria by the cytochrome P450 CYP112 in three individual steps, which involves large structural changes in the transition from substrate to product, with further divergence in the recently demonstrated use of two separate mechanistic routes...
July 5, 2018: Biochemical Journal
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