Home  About us  Contact
 

Cofactor Binding (cofactor + binding)

Distribution by Scientific Domains
Chemistry100%

Terms modified by Cofactor Binding

  • cofactor binding site
    		•

    Selected Abstracts

    Functional Characterization of the Recombinant N -Methyltransferase Domain from the Multienzyme Enniatin Synthetase

    CHEMBIOCHEM, Issue 9 2007
    Till Hornbogen Dr.
    Abstract A 51 kDa fusion protein incorporating the N -methyltransferase domain of the multienzyme enniatin synthetase from Fusarium scirpi was expressed in Saccharomyces cerevisiae. The protein was purified and found to bind S -adenosyl methionine (AdoMet) as demonstrated by cross-linking experiments with 14C-methyl-AdoMet under UV irradiation. Cofactor binding at equilibrium conditions was followed by saturation transfer difference (STD) NMR spectroscopy, and the native conformation of the methyltransferase was assigned. STD NMR spectroscopy yielded significant signals for H2 and H8 of the adenine moiety, H1' of D -ribose, and SCH3 group of AdoMet. Methyl group transfer catalyzed by the enzyme was demonstrated by using aminoacyl- N -acetylcysteamine thioesters (aminoacyl-SNACs) of L -Val, L -Ile, and L -Leu, which mimic the natural substrate amino acids of enniatin synthetase presented by the enzyme bound 4,-phosphopantetheine arm. In these experiments the enzyme was incubated in the presence of the corresponding aminoacyl-SNAC and 14C-methyl-AdoMet for various lengths of time, for up to 30 min. N -[14C-Methyl]-aminoacyl-SNAC products were extracted with EtOAc and separated by TLC. Acid hydrolysis of the isolated labeled compounds yielded the corresponding N -[14C-methyl] amino acids. Further proof for the formation of N - 14C-methyl-aminoacyl-SNACs came from MALDI-TOF mass spectrometry which yielded 23,212 Da for N -methyl-valyl-SNAC, accompanied by the expected postsource decay (PSD) pattern. Interestingly, L -Phe, which is not a substrate amino acid of enniatin synthetase, also proved to be a methyl group acceptor. D -Val was not accepted as a substrate; this indicates selectivity for the L isomer. [source]

    Evolutionary divergence of valosin-containing protein/cell division cycle protein 48 binding interactions among endoplasmic reticulum-associated degradation proteins

    FEBS JOURNAL, Issue 5 2009
    Giacomo Morreale
    Endoplasmic reticulum (ER)-associated degradation (ERAD) is a cell-autonomous process that eliminates large quantities of misfolded, newly synthesized protein, and is thus essential for the survival of any basic eukaryotic cell. Accordingly, the proteins involved and their interaction partners are well conserved from yeast to mammals, and Saccharomyces cerevisiae is widely used as a model system with which to investigate this fundamental cellular process. For example, valosin-containing protein (VCP) and its yeast homologue cell division cycle protein 48 (Cdc48p), which help to direct polyubiquitinated proteins for proteasome-mediated degradation, interact with an equivalent group of ubiquitin ligases in mouse and in S. cerevisiae. A conserved structural motif for cofactor binding would therefore be expected. We report a VCP-binding motif (VBM) shared by mammalian ubiquitin ligase E4b (Ube4b),ubiquitin fusion degradation protein 2a (Ufd2a), hydroxymethylglutaryl reductase degradation protein 1 (Hrd1),synoviolin and ataxin 3, and a related sequence in Mr 78 000 glycoprotein,Amfr with slightly different binding properties, and show that Ube4b and Hrd1 compete for binding to the N-terminal domain of VCP. Each of these proteins is involved in ERAD, but none has an S. cerevisiae homologue containing the VBM. Some other invertebrate model organisms also lack the VBM in one or more of these proteins, in contrast to vertebrates, where the VBM is widely conserved. Thus, consistent with their importance in ERAD, evolution has developed at least two ways to bring these proteins together with VCP,Cdc48p. However, the differing molecular architecture of VCP,Cdc48p complexes indicates a key point of divergence in the molecular details of ERAD mechanisms. [source]

    Importance of tyrosine residues of Bacillus stearothermophilus serine hydroxymethyltransferase in cofactor binding and l - allo -Thr cleavage

    FEBS JOURNAL, Issue 18 2008
    Crystal structure, biochemical studies
    Serine hydroxymethyltransferase (SHMT) from Bacillus stearothermophilus (bsSHMT) is a pyridoxal 5,-phosphate-dependent enzyme that catalyses the conversion of l -serine and tetrahydrofolate to glycine and 5,10-methylene tetrahydrofolate. In addition, the enzyme catalyses the tetrahydrofolate-independent cleavage of 3-hydroxy amino acids and transamination. In this article, we have examined the mechanism of the tetrahydrofolate-independent cleavage of 3-hydroxy amino acids by SHMT. The three-dimensional structure and biochemical properties of Y51F and Y61A bsSHMTs and their complexes with substrates, especially l - allo -Thr, show that the cleavage of 3-hydroxy amino acids could proceed via C, proton abstraction rather than hydroxyl proton removal. Both mutations result in a complete loss of tetrahydrofolate-dependent and tetrahydrofolate-independent activities. The mutation of Y51 to F strongly affects the binding of pyridoxal 5,-phosphate, possibly as a consequence of a change in the orientation of the phenyl ring in Y51F bsSHMT. The mutant enzyme could be completely reconstituted with pyridoxal 5,-phosphate. However, there was an alteration in the ,max value of the internal aldimine (396 nm), a decrease in the rate of reduction with NaCNBH3 and a loss of the intermediate in the interaction with methoxyamine (MA). The mutation of Y61 to A results in the loss of interaction with C, and C, of the substrates. X-Ray structure and visible CD studies show that the mutant is capable of forming an external aldimine. However, the formation of the quinonoid intermediate is hindered. It is suggested that Y61 is involved in the abstraction of the C, proton from 3-hydroxy amino acids. A new mechanism for the cleavage of 3-hydroxy amino acids via C, proton abstraction by SHMT is proposed. [source]

    De novo proteins from designed combinatorial libraries

    PROTEIN SCIENCE, Issue 7 2004
    Michael H. Hecht
    Abstract Combinatorial libraries of de novo amino acid sequences can provide a rich source of diversity for the discovery of novel proteins with interesting and important activities. Randomly generated sequences, however, rarely fold into well-ordered proteinlike structures. To enhance the quality of a library, features of rational design must be used to focus sequence diversity into those regions of sequence space that are most likely to yield folded structures. This review describes how focused libraries can be constructed by designing the binary pattern of polar and nonpolar amino acids to favor proteins that contain abundant secondary structure, while simultaneously burying hydrophobic side chains and exposing hydrophilic side chains to solvent. The "binary code" for protein design was used to construct several libraries of de novo proteins, including both ,-helical and ,-sheet structures. The recently determined solution structure of a binary patterned four-helix bundle is well ordered, thereby demonstrating that sequences that have neither been selected by evolution (in vivo or in vitro) nor designed by computer can form nativelike proteins. Examples are presented demonstrating how binary patterned libraries have successfully produced well-ordered structures, cofactor binding, catalytic activity, self-assembled monolayers, amyloid-like nanofibrils, and protein-based biomaterials. [source]

    Expansion of the aspartate ,-semialdehyde dehydrogenase family: the first structure of a fungal ortholog

    ACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2010
    Buenafe T. Arachea
    The enzyme aspartate semialdehyde dehydrogenase (ASADH) catalyzes a critical transformation that produces the first branch-point intermediate in an essential microbial amino-acid biosynthetic pathway. The first structure of an ASADH isolated from a fungal species (Candida albicans) has been determined as a complex with its pyridine nucleotide cofactor. This enzyme is a functional dimer, with a similar overall fold and domain organization to the structurally characterized bacterial ASADHs. However, there are differences in the secondary-structural elements and in cofactor binding that are likely to cause the lower catalytic efficiency of this fungal enzyme. Alterations in the dimer interface, through deletion of a helical subdomain and replacement of amino acids that participate in a hydrogen-bonding network, interrupt the intersubunit-communication channels required to support an alternating-site catalytic mechanism. The detailed functional information derived from this new structure will allow an assessment of ASADH as a possible target for antifungal drug development. [source]

    Structures of the apo and holo forms of formate dehydrogenase from the bacterium Moraxella sp.

    ACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2009
    C-1: towards understanding the mechanism of the closure of the interdomain cleft
    NAD+ -dependent formate dehydrogenase (FDH) catalyzes the oxidation of formate ion to carbon dioxide coupled with the reduction of NAD+ to NADH. The crystal structures of the apo and holo forms of FDH from the methylotrophic bacterium Moraxella sp. C-1 (MorFDH) are reported at 1.96 and 1.95,Å resolution, respectively. MorFDH is similar to the previously studied FDH from the bacterium Pseudomonas sp. 101 in overall structure, cofactor-binding mode and active-site architecture, but differs in that the eight-residue-longer C-terminal fragment is visible in the electron-density maps of MorFDH. MorFDH also differs in the organization of the dimer interface. The holo MorFDH structure supports the earlier hypothesis that the catalytic residue His332 can form a hydrogen bond to both the substrate and the transition state. Apo MorFDH has a closed conformation of the interdomain cleft, which is unique for an apo form of an NAD+ -dependent dehydrogenase. A comparison of the structures of bacterial FDH in open and closed conformations allows the differentiation of the conformational changes associated with cofactor binding and domain motion and provides insights into the mechanism of the closure of the interdomain cleft in FDH. The C-terminal residues 374,399 and the substrate (formate ion) or inhibitor (azide ion) binding are shown to play an essential role in the transition from the open to the closed conformation. [source]

    Active-site changes in the pyruvate dehydrogenase multienzyme complex E1 apoenzyme component from Escherichia coli observed at 2.32,Å resolution

    ACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2006
    Palaniappa Arjunan
    The first enzymatic component, E1 (EC 1.2.4.1), of the pyruvate dehydrogenase multienzyme complex (PDHc) utilizes thiamine diphosphate (ThDP) and Mg2+ as cofactors. The structure of a branched-chain-specific E1 apoenzyme from the heterotetrameric ,2,2 E1 family was recently reported and showed that disorder-to-order transformations in two active-site loops take place upon cofactor binding. To ascertain what effect the absence of cofactor may have in the homodimeric ,2Escherichia coli PDHc E1, the corresponding apoenzyme has been prepared and its three-dimensional structure determined and analyzed at 2.32,Å by crystallographic methods. This represents the first reported apoenzyme structure for any E1 component from the homodimeric ,2 family. Electron-density features occurring in the region where the cofactor pyrimidine ring would normally be expected to bind are of size, shape and location compatible with water molecules that form a hydrogen-bonded linkage between residues Glu571 and Val192, which normally make conserved interactions with the ThDP cofactor. A histidine side chain that normally forms hydrogen bonds to ThDP is disordered in its absence and partially occupies two sites. Unlike in the reported heterotetrameric branched-chain apo-­E1, no disorder/order loop transformations are evident in apo-PDHc E1 relative to the holo-E1 enzyme (PDHc E1,ThDP,Mg2+). Differences in the extent of hydrogen-bonding networks found in the apo-E1 enzyme, the holo-E1 enzyme and in an inhibitor complex with bound thiamine 2-thiazolone diphosphate (ThTDP), PDHc E1,ThTDP,Mg2+, are described. [source]