J. Van Beeumen

Ampd, Essential for Both Beta-Lactamase Regulation and Cell Wall Recycling, Is a Novel Cytosolic N-Acetylmuramyl-L-Alanine Amidase

In enterobacteria, the ampD gene encodes a cytosolic protein which acts as a negative regulator of β‐lactamase expression. It is shown here that the AmpD protein is a novel N‐acetylmuramyl‐L‐alanine amidase (E.C.3.5.1.28) participating in the intracellular recycling of peptido‐glycan fragments. Surprisingly, AmpD exhibits an exclusive specificity for substrates containing anhydro muramic acid. This anhydro bond is mainly found in the peptidoglycan degradation products formed by the periplasmic lytic transglycosylases and thus might behave as a‘recycling tag’allowing the enzyme to distinguish these fragments from the newly synthesized peptidoglycan precursors. The AmpD substrate (or substrates) which accumulates in the absence of the corresponding enzymatic activity acts as an intracellular positive effector for β‐lactamase expression and might represent an element of a communication network between the chromosome and the cell wall peptidoglycan.

Primary structure and processing of lysosomal alpha-glucosidase; homology with the intestinal sucrase-isomaltase complex.

Lysosomal alpha‐glucosidase (acid maltase) is essential for degradation of glycogen in lysosomes. Enzyme deficiency results in glycogenosis type II. The amino acid sequence of the entire enzyme was derived from the nucleotide sequence of cloned cDNA. The cDNA comprises 3636 nt, and hybridizes with a messenger RNA of approximately 3.6 kb, which is absent in fibroblasts of two patients with glycogenosis type II. The encoded protein has a molecular mass of 104.645 kd and starts with a signal peptide. Sites of proteolytic processing are established by identification of N‐terminal amino acid sequences of the 110‐kd precursor, and the 76‐kd and 70‐kd mature forms of the enzyme encoded by the cDNA. Interestingly, both amino‐terminal and carboxy‐terminal processing occurs. Sites of sugar‐chain attachment are proposed. A remarkable homology is observed between this soluble lysosomal alpha‐glucosidase and the membrane‐bound intestinal brush border sucrase‐isomaltase enzyme complex. It is proposed that these enzymes are derived from the same ancestral gene. Around the putative active site of sucrase and isomaltase, 10 out of 13 amino acids are identical to the corresponding amino acids of lysosomal alpha‐glucosidase. This strongly suggests that the aspartic acid residue at this position is essential for catalytic function of lysosomal alpha‐glucosidase.

Characterization of a bacterial tyrosine ammonia lyase, a biosynthetic enzyme for the photoactive yellow protein

During genome sequence analysis of Rhodobacter capsulatus, nearby open reading frames were found that encode a photoactive yellow protein (PYP) and a hypothetical biosynthetic enzyme for its chromophore, a tyrosine ammonia lyase (TAL). We isolated the TAL gene, overproduced the recombinant protein in Escherichia coli, and after purification analyzed the enzyme for its activity. The catalytic efficiency for tyrosine was shown to be approximately 150 times larger than for phenylalanine, suggesting that the enzyme could in fact be involved in biosynthesis of the PYP chromophore. To our knowledge it is the first time this type of enzyme has been found in bacteria.

The Structure of a Cold-Adapted Family 8 Xylanase at 1.3 A Resolution: Structural Adaptations to Cold and Investigation of the Active Site

Enzymes from psychrophilic organisms differ from their mesophilic counterparts in having a lower thermostability and a higher specific activity at low and moderate temperatures. The current consensus is that they have an increased flexibility, enhancing accommodation and transformation of the substrates at low energy costs. Here we describe the structure of the xylanase from the Antarctic bacterium Pseudoalteromonas haloplanktis at 1.3 Å resolution. Xylanases are usually grouped into glycosyl hydrolase families 10 and 11, but this enzyme belongs to family 8. The fold differs from that of other known xylanases and can be described as an (α/α)6 barrel. Various parameters that may explain the cold-adapted properties were examined and indicated that the protein has a reduced number of salt bridges and an increased exposure of hydrophobic residues. The crystal structures of a complex with xylobiose and of mutant D144N were obtained at 1.2 and 1.5 Å resolution, respectively. Analysis of the various substrate binding sites shows that the +3 and −3 subsites are rearranged as compared to those of a family 8 homolog, while the xylobiose complex suggests the existence of a +4 subsite. A decreased acidity of the substrate binding cleft and an increased flexibility of aromatic residues lining the subsites may enhance the rate at which substrate is bound.

Cross-linking of ubiquitin, HSP27, parkin, and α-synuclein by γ-glutamyl-ε-lysine bonds in Alzheimer's neurofibrillary tangles

The accumulation of misfolded proteins in intracellular inclusions is a generic feature of neurodegenerative disorders. Although heavily ubiquitylated, the aggregated proteins are not degraded by the proteasomes. A possible reason for this phenomenon may be a modification of deposited proteins by transglutaminases forming γ‐glutamyl‐ε‐lysine (GGEL) cross‐links between distinct proteins. Here, we show that the frequency of GGEL cross‐links is an order of magnitude higher in Alzheimers brain cortex than in age‐matched or younger controls. This difference is due to the accumulation of GGEL cross‐links in ubiquitin‐immunopositive protein particles present in both Alzheimers brains and those from aged individuals. The highly cross‐ linked protein aggregates show immunoreactivity to antibodies against tau and neurofilament proteins, and partially also to α‐synuclein, indicating that these structures are inherent in Alzheimers neurofibrillary tangles and Lewy bodies. Using mass sequence analysis, we identified the same six pairs of peptide sequences cross‐linked in both senile and Alzheimers specimens: Gln31 and Gln190 of HSP27 protein are cross‐linked with Lys29 and Lys48 of ubiquitin and HSP27 therefore may cross‐link two (poly)ubiquitin chains. One lysine residue of parkin and one of α‐synuclein were also found to be cross‐linked. The data suggest that cross‐linking of (poly)ubiquitin moieties via HSP27 may have a role in the stabilization of the intraneuronal protein aggregates by interference with the proteasomal elimination of unfolded proteins.

The Xanthopsins: a new family of eubacterial blue-light photoreceptors

Photoactive yellow protein (PYP) is a photoreceptor that has been isolated from three halophilic phototrophic purple bacteria. The PYP from Ectothiorhodospira halophila BN9626 is the only member for which the sequence has been reported at the DNA level. Here we describe the cloning and sequencing of the genes encoding the PYPs from E.halophila SL‐1 (type strain) and Rhodospirillum salexigens. The latter protein contains, like the E.halophila PYP, the chromophore trans p‐coumaric acid, as we show here with high performance capillary zone electrophoresis. Additionally, we present evidence for the presence of a gene encoding a PYP homolog in Rhodobacter sphaeroides, the first genetically well‐characterized bacterium in which this photoreceptor has been identified. An ORF downstream of the pyp gene from E.halophila encodes an enzyme, which is proposed to be involved in the biosynthesis of the chromophore of PYP. The pyp gene from E.halophila was used for heterologous overexpression in both Escherichia coli and R.sphaeroides, aimed at the development of a holoPYP overexpression system (an intact PYP, containing the p‐coumaric acid chromophore and displaying the 446 nm absorbance band). In both organisms the protein could be detected immunologically, but its yellow color was not observed. Molecular genetic construction of a histidine‐tagged version of PYP led to its 2500‐fold overproduction in E.coli and simplified purification of the heterologously produced apoprotein. HoloPYP could be reconstituted by the addition of p‐coumaric anhydride to the histidine‐tagged apoPYP (PYP lacking its chromophore). We propose to call the family of photoactive yellow proteins the xanthopsins, in analogy with the rhodopsins.

Two tandemly linked identical genes code for the glycosomal glyceraldehyde-phosphate dehydrogenase in Trypanosoma brucei.

Trypanosoma brucei contains two isoenzymes for glyceraldehyde‐phosphate dehydrogenase (GAPDH); one enzyme resides in a microbody‐like organelle, the glycosome, the other one is found in the cytosol. We show here that the glycosomal enzyme is encoded by two tandemly linked genes of identical sequence. These genes code for a protein of 358 amino acids, with a mol. wt of 38.9 kd. This is considerably larger than all other GAPDH proteins studied so far, including the enzyme that is located in the cytosol of the trypanosome. The glycosomal enzyme shows 52‐57% homology with known sequences of GAPDH proteins from 10 other organisms, both prokaryotes and eukaryotes. The residues that are involved in NAD+ binding, catalysis and subunit contacts are well conserved between all these GAPDH molecules, including the trypanosomal one. However, the glycosomal protein of T. brucei has some distinct features. Firstly, it contains a number of insertions, 1‐8 amino acids long, which are responsible for the high mol. wt of the protein. Secondly, an unusually high number of positively charged amino acids confer a high isoelectric point (pI 9.3) to the protein. Part of the additional basic residues are present in the insertions. We discuss the genomic organization of the genes for the glycosomal GAPDH and the possibility that the particular features of the protein are involved in its transfer from the cytoplasm, where it is synthesized, into the glycosome.

Common elements on the surface of glycolytic enzymes from Trypanosoma brucei may serve as topogenic signals for import into glycosomes.

In Trypanosoma brucei, a major pathogenic protozoan parasite of Central Africa, a number of glycolytic enzymes present in the cytosol of other organisms are uniquely segregated in a microbody‐like organelle, the glycosome, which they are believed to reach post‐translationally after being synthesized by free ribosomes in the cytosol. In a search for possible topogenic signals responsible for import into glycosomes we have compared the amino acid sequences of four glycosomal enzymes: triosephosphate isomerase (TIM), glyceraldehyde‐phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK) and aldolase (ALDO), with each other and with their cytosolic counterparts. Each of these enzymes contains a marked excess of positive charges, distributed in two or more clusters along the polypeptide chain. Modelling of the three‐dimensional structures of TIM, PGK and GAPDH using the known structural coordinates of homologous enzymes from other organisms indicates that all three may have in common two ‘hot spots’ about 40 A apart, which themselves include a pair of basic amino acid residues separated by a distance of about 7 A. The sequence of glycosomal ALDO, for which no three‐dimensional information is available, is compatible with the presence of the same configuration on the surface of this enzyme. We propose that this feature plays an essential role in the import of enzymes into glycosomes.

Microbial Reduction and Precipitation of Vanadium by Shewanella oneidensis

ABSTRACT Shewanella oneidensis couples anaerobic oxidation of lactate, formate, and pyruvate to the reduction of vanadium pentoxide (VV). The bacterium reduces VV (vanadate ion) to VIV (vanadyl ion) in an anaerobic atmosphere. The resulting vanadyl ion precipitates as a VIV-containing solid.

Biochemical characterization and mode of action of a thermostable endoglucanase purified from Thermoascus aurantiacus

A major extracellular endoglucanase purified to homogeneity from Thermoascus aurantiacus had a M(r) of 34 kDa and a pI of 3.7 and was optimally active at 70-80 degrees C and pH 4.0-4.4. It was stable at pH 2.8-6.8 at 50 degrees C for 48 h and maintained its secondary structure and folded conformation up to 70 degrees C at pH 5.0 and 2.8, respectively. A 33-amino acid sequence at the N terminus showed considerable homology with 14 microbial endoglucanases having highly conserved 8 amino acids (positions 10-17) and Gly, Pro, Gly, and Pro at positions 8, 22, 23, and 32, respectively. The enzyme is rich in Asp (15%) and Glu (10%) with a carbohydrate content of 2.7%. Polyclonal antibodies of endoglucanase cross-reacted with their own antigen and with other purified cellulases from T. aurantiacus. The endoglucanase was specific for polymeric substrates with highest activity toward carboxymethyl cellulose followed by barley beta-glucan and lichenan. It preferentially cleaved the internal glycosidic bonds of Glc(n) and MeUmbGlc(n) and possessed an extended substrate-binding site with five subsites. The data indicate that the endoglucanase from T. aurantiacus is a member of glycoside hydrolase family 5.