Febs Letters

doi: 10.1016/S0014-5793(02)02735-7pmid: N/A

Vafai, Scott B; Stock, Jeffry B

doi: 10.1016/S0014-5793(02)02702-3pmid: 11997007

Tau hyperphosphorylation is a central event in the development of Alzheimer's Disease (AD). Protein phosphatase 2A (PP2A) heterotrimer formation is necessary for efficient dephosphorylation of the tau protein. S‐Adenosylmethionine‐dependent carboxyl methylation is essential for the assembly of PP2A heterotrimers. Epidemiological evidence indicates that elevated plasma homocysteine is an independent risk factor for AD. Homocysteine is a key intermediate in the methyl cycle and elevated plasma homocysteine results in a global decrease in cellular methylation. We propose that the PP2A methylation system is the link relating elevated plasma homocysteine to AD.

Berglund, Magnus M; Fredriksson, Robert; Salaneck, Erik; Larhammar, Dan

doi: 10.1016/S0014-5793(02)02534-6pmid: 11997008

The neuropeptide Y (NPY) receptor Y2 antagonist BIIE0246 has sub‐nanomolar affinity for the human Y2 (hY2) receptor but binds very poorly to chicken Y2 (chY2) with micromolar affinity. Sequence comparisons identified several amino acids for investigation by mutagenesis. Reciprocal mutagenesis between hY2 and chY2 revealed that three of these, individually and in combination, are important for BIIE0246 binding, namely positions Gln135 in transmembrane (TM) 3, Leu227 in TM5, and Leu284 in TM6. Mutagenesis of hY2 to the corresponding amino in chY2 (generating hY2[Q135H,L227Q,L284F]) made the affinity of BIIE0246 as low as for chY2. Introduction into chY2 of the three human residues resulted in antagonist affinity almost as high as for hY2. To distinguish between direct and indirect effects, each of the three residues in hY2 was replaced with alanine. BIIE0246 bound with 28‐fold lower affinity to hY2[L227A], suggesting the Leu227 interacts directly with the antagonist. The other two alanine mutants bound with unaltered affinity, suggesting that the corresponding chY2 residues abolish binding through steric hindrance or charge repulsion. Thus, three amino acid residues can in an additive manner completely account for the difference in antagonist binding between the hY2 and chY2 receptors. These results will be useful for construction of three‐dimensional models of the widely divergent NPY receptor subtypes.

McEwan, Alastair G; Lewin, Allison; Davy, Sharon L; Boetzel, Ruth; Leech, Andrew; Walker, Daniel; Wood, Tania; Moore, Geoffrey R

doi: 10.1016/S0014-5793(02)02532-2pmid: 11997009

PrrC from Rhodobacter sphaeroides provides the signal input to a two‐component signal transduction system that senses changes in oxygen tension and regulates expression of genes involved in photosynthesis (Eraso, J.M. and Kaplan, S. (2000) Biochemistry 39, 2052‐2062; Oh, J.‐I. and Kaplan, S. (2000) EMBO J. 19, 4237‐4247). It is also a homologue of eukaryotic Sco proteins and each has a C‐x‐x‐x‐C‐P sequence. In mitochondrial Sco proteins these cysteines appear to be essential for the biogenesis of the CuA centre of respiratory cytochrome oxidase. Overexpression and purification of a water‐soluble and monomeric form of PrrC has provided sufficient material for a chemical and spectroscopic study of the properties of the four cysteine residues of PrrC, and its ability to bind divalent cations, including copper. PrrC expressed in the cytoplasm of Escherichia coli binds Ni2+ tightly and the data are consistent with a mononuclear metal site. Following removal of Ni2+ and formation of renatured metal‐free rPrrC (apo‐PrrC), Cu2+ could be loaded into the reduced form of PrrC to generate a protein with a distinctive UV‐visible spectrum, having absorbance with a λ max of 360 nm. The copper:PrrC ratio is consistent with the presence of a mononuclear metal centre. The cysteines of metal‐free PrrC oxidise in the presence of air to form two intramolecular disulfide bonds, with one pair being extremely reactive. The cysteine thiols with extreme O2 sensitivity are involved in copper binding in reduced PrrC since the same copper‐loaded protein could not be generated using oxidised PrrC. Thus, it appears that PrrC, and probably Sco proteins in general, could have both a thiol‐disulfide oxidoreductase function and a copper‐binding role.

Tamulaitis, Gintautas; Solonin, Alexander S.; Siksnys, Virginijus

doi: 10.1016/S0014-5793(02)02621-2pmid: 11997010

A catalytic sequence motif PDX10–30(E/D)XK is found in many restriction enzymes. On the basis of sequence similarities and mapping of the conserved residues to the crystal structure of NgoMIV we suggest that residues D160, K182, R186, R188 and E195 contribute to the catalytic/DNA binding site of the Ecl18kI restriction endonuclease. Mutational analysis confirms the functional significance of the conserved residues of Ecl18kI. Therefore, we conclude that the active site motif 159VDX21KX12E of Ecl18kI differs from the canonical PDX10–30(E/D)XK motif characteristic for most of the restriction enzymes. Moreover, we propose that two subfamilies of endonucleases Ecl18kI/PspGI/EcoRII and Cfr10I/Bse634I/NgoMIV, specific, respectively, for CCNGG/CCWGG and RCCGGY/GCCGGC sites, share conserved active site architecture and DNA binding elements.

Schmitt, Anja; Gutierrez, Gustavo J; Lénárt, Péter; Ellenberg, Jan; Nebreda, Angel R

doi: 10.1016/S0014-5793(02)02630-3pmid: 11997011

Here we show that during the meiotic maturation of Xenopus oocytes, histone H3 becomes phosphorylated on serine‐10 at about the time of maturation promoting factor activation and meiosis I entry. However, overexpression of cAMP‐dependent protein kinase that blocks entry into M phase, also leads to massive serine‐10 phosphorylation of histone H3 in intact Xenopus oocytes but does not cause chromosome condensation. We also show that the phosphorylation of histone H3 during oocyte maturation requires the activation of the mitogen‐activated protein kinase/p90Rsk pathway. Our results indicate that in G2‐arrested oocytes, which are about to enter M phase, histone H3 phosphorylation is not sufficient for chromosome condensation.

Mason, Maria G.; Wilson, Michael T.; Ball, Andrew; Nicholls, Peter

doi: 10.1016/S0014-5793(02)02633-9pmid: 11997012

We have used optical and electron paramagnetic spectroscopy to study the flavohaem enzyme cellobiose oxidoreductase (CBOR) from Phanerochaete chrysosporium. We have examined redox cycles of the enzyme in which the oxidation of cellobiose to cellobionolactone is coupled to the reduction of oxygen. During turnover flavin can reduce oxygen with one electron to produce superoxide or two electrons to produce hydrogen peroxide. Addition of superoxide dismutase significantly extended the time courses of these cycles, slowing the re‐oxidation rate of both cofactors. Addition of catalase also affected the haem time course, but to a lesser extent. Experiments in which superoxide was generated in the reaction mixture showed that this radical greatly enhanced the rate of haem re‐oxidation. From these results we propose a mechanism in which reactive oxygen species generation by CBOR flavin subsequently re‐oxidises CBOR haem. We discuss this mechanism in relationship to the biological function of this enzyme, namely lignocellulose degradation.

Hemmi, Hikaru; Ishibashi, Jun; Hara, Seiichi; Yamakawa, Minoru

doi: 10.1016/S0014-5793(02)02637-6pmid: 11997013

A novel antibacterial peptide, moricin, isolated from the silkworm Bombyx mori, consists of 42 amino acids. It is highly basic and the amino acid sequence has no significant similarity to those of other antibacterial peptides. The 20 structures of moricin in methanol have been determined from two‐dimensional 1H‐nuclear magnetic resonance spectroscopic data. The solution structure reveals an unique structure comprising of a long α‐helix containing eight turns along nearly the full length of the peptide except for four N‐terminal residues and six C‐terminal residues. The electrostatic surface map shows that the N‐terminal segment of the α‐helix, residues 5–22, is an amphipathic α‐helix with a clear separation of hydrophobic and hydrophilic faces, and that the C‐terminal segment of the α‐helix, residues 23–36, is a hydrophobic α‐helix except for the negatively charged surface at the position of Asp30. The results suggest that the amphipathic N‐terminal segment of the α‐helix is mainly responsible for the increase in permeability of the membrane to kill the bacteria.

Kursula, Inari; Partanen, Sanna; Lambeir, Anne-Marie; Wierenga, Rik K.

doi: 10.1016/S0014-5793(02)02639-Xpmid: 11997014

Triosephosphate isomerase (TIM) has a conserved salt bridge 20 Å away from both the active site and the dimer interface. In this study, four salt bridge mutants of Trypanosoma brucei brucei TIM were characterized. The folding and stability of the mutants are impaired compared to the wild‐type enzyme. This salt bridge is part of a hydrogen bonding network which tethers the C‐terminal β7α7β8α8 unit to the bulk of the protein. In the variants D227N, D227A, and R191S, this network is preserved, as can be deduced from the structure of the R191S variant. In the R191A variant, the side chain at position 191 cannot contribute to this network. Also the catalytic power of this variant is most affected.

Fraaije, Marco W.; Kamerbeek, Nanne M.; van Berkel, Willem J.H.; Janssen, Dick B.

doi: 10.1016/S0014-5793(02)02623-6pmid: 11997015

Baeyer–Villiger monooxygenases (BVMOs) form a distinct class of flavoproteins that catalyze the insertion of an oxygen atom in a C–C bond using dioxygen and NAD(P)H. Using newly characterized BVMO sequences, we have uncovered a BVMO‐identifying sequence motif: FXGXXXHXXXW(P/D). Studies with site‐directed mutants of 4‐hydroxyacetophenone monooxygenase from Pseudomonas fluorescens ACB suggest that this fingerprint sequence is critically involved in catalysis. Further sequence analysis showed that the BVMOs belong to a novel superfamily that comprises three known classes of FAD‐dependent monooxygenases: the so‐called flavin‐containing monooxygenases (FMOs), the N‐hydroxylating monooxygenases (NMOs), and the BVMOs. Interestingly, FMOs contain an almost identical sequence motif when compared to the BVMO sequences: FXGXXXHXXX(Y/F). Using these novel amino acid sequence fingerprints, BVMOs and FMOs can be readily identified in the protein sequence databank.