The ENZYME data bank in 1999

The ENZYME data bank is a repository of information related to the nomenclature of enzymes. In recent years it has become an indispensable resource for the development of metabolic databases. The current version contains information on 3704 enzymes. It is available through the ExPASy WWW server (http://www.expasy.ch/

Proteomic analysis of Plasmodium in the mosquito: progress and pitfalls

Here we discuss proteomic analyses of whole cell preparations of the mosquito stages of malaria parasite development (i.e. gametocytes, microgamete, ookinete, oocyst and sporozoite) of Plasmodium berghei. We also include critiques of the proteomes of two cell fractions from the purified ookinete, namely the micronemes and cell surface. Whereas we summarise key biological interpretations of the data, we also try to identify key methodological constraints we have met, only some of which we were able to resolve. Recognising the need to translate the potential of current genome sequencing into functional understanding, we report our efforts to develop more powerful combinations of methods for the in silico prediction of protein function and location. We have applied this analysis to the proteome of the male gamete, a cell whose very simple structural organisation facilitated interpretation of data. Some of the in silico predictions made have now been supported by ongoing protein tagging and genetic knockout studies. We hope this discussion may assist future studie

Practical Use of High-level Petri Nets

This booklet contains the proceedings of the Workshop on Practical Use of High-level Petri Nets, June 27, 2000. The workshop is part of the 21st International Conference on Application and Theory of Petri Nets organised by the CPN group at the Department of Computer Science, University of Aarhus, Denmark. The workshop papers are available in electronic form via the web pages: http://www.daimi.au.dk/pn2000/proceeding

Practical Use of High-level Petri Nets

This booklet contains the proceedings of the Workshop on Practical Use of High-level Petri Nets, June 27, 2000. The workshop is part of the 21st International Conference on Application and Theory of Petri Nets organised by the CPN group at the Department of Computer Science, University of Aarhus, Denmark. The workshop papers are available in electronic form via the web pages: http://www.daimi.au.dk/pn2000/proceeding

Thermophilic mixed culture degradation of Miscanthus x giganteus as a guide to strategies for consolidated bioprocessing

The successful development of consolidated bioprocessing requires microorganisms capable of degrading lignocellulosic biomass and fermenting the resulting sugars. Commercial cellulases and hemicellulases are currently being used to access these sugars, adding to the cost of producing useful products from lignocellulose. This study reports the enrichment of thermophilic, miscanthus degrading bacterial cultures from a municipal composting facility. The detected and isolated bacteria were characterized by 16S rRNA gene sequence analysis and were mostly Chitinophagaceae family, Meiothermus spp. and Geobacillus spp. Other isolated species included Cohnella spp., Brevibacillus sp., Chelatococcus spp., Thermobacillus spp., Thermoanaerobacterium spp., Thermobispora bispora, Bacillus spp., Staphylococcus sp. and Micrococcus sp. After enrichment, the mixed population was able to degrade greater than 50% of an ammonium hydroxide pre-treated Miscanthus x giganteus sample (1 g) over a six week incubation period at 55oC, with a reduction in the amounts of all components, including acid soluble and acid insoluble lignin. The glycoside hydrolases and other enzymes identified in the culture supernatants included endo-1,4-β-glucanase A, glucoamylase, xylan 1,4-β-xylosidase, xylose isomerase, xylulokinase, superoxide dismutase, transaldolase, Mn-catalase, Δ-1-pyrroline-5-carboxylate dehydrogenase and endo-β-N-acetylglucoseaminidase H. The HPLC analysis showed that fermentation products formate and lactate were present in the culture supernatant. Expression of an endoglycoside hydrolase (Csac_0137 from Caldicellulosiruptor saccharolyticus) gene in Geobacillus thermoglucosidasius strains, NCIMB 11955 and DL33, improved their β-glucosidase specific activity on cellobiose, and improved glycoside hydrolase activities of recombinant DL33 strain when grown on pre-treated M. x giganteus. Co-culturing of either transformed or wild-type NCIMB 11955 and DL33 with some of the isolated strains improved their glycoside hydrolase activity and growth on pretreated M. x giganteus.Open Acces

A conserved Inner Membrane Protein of Aggregatibacter actinomycetemcomitans is integral for membrane function

The cell envelope of Aggregatibacter actinomycetemcomitans, a Gram-negative pathogenic bacterium implicated in human oral and systemic disease, plays a critical role in maintenance of cellular homeostasis, resistance to external stress, and host\u27pathogen interactions. Our laboratory has identified a novel gene product, morphogenesis protein C (MorC), deletion of which leads to multiple pleotropic effects pertaining to membrane structure and function. The MorC sequence was determined to be conserved in Gammaproteobacteria. Based on this bioinformatic analysis, the functional conservation of this protein was investigated utilizing an A. actinomycetemcomitans morC mutant as a model system to express homologs from four phylogenetically diverse representatives of the Gammaproteobacteria: Haemophilus influenzae, Escherichia coli, Pseudomonas aeruginosa, and Moraxella catarrhalis. MorC from all organisms restored at least one of the A. actinomycetemcomitans mutant phenotypes, implying that the protein is functionally conserved across Gammaproteobacteria. Further, deletion mutagenesis indicated that the last 10 amino acids of the carboxyl terminus were necessary to maintain the integrity of the membrane. The observed pleiotropic effects suggested alterations in the membrane protein composition of the morC mutant. Stable isotope dimethyl labeling in conjunction with mass spectrometry was employed to quantitatively determine the differences in the abundance of membrane proteins of the isogenic mutant and wild-type strains. A total of 665 envelope associated proteins were identified and functionally annotated using bioinformatic tools. All proteins, except MorC, were detected in the mutant strain. However, 12 proteins were found in lesser (10) or greater (2) abundance in the membrane preparation of the mutant strain. These proteins were ascribed functions associated with protein quality control, oxidative stress response, and protein secretion systems. One protein found to be reduced was a component of the fimbrial secretion system of A. actinomycetemcomitans. The significance of this finding was unclear due to the afimbriated nature of the laboratory strain used in the study. Therefore, the defect in fimbriation was identified and complemented in trans. The transformed strain displayed all of the hallmarks of a naturally fimbriated strain including: a distinct star-like colony morphology; robust biofilm formation; and presence of fimbriae as detected by electron microscopy. The isogenic morC mutant strain transformed with an identical plasmid did not display any fimbriated phenotypes. The role of MorC in fimbriae production of a naturally fimbriated strain was investigated by inactivation of morC in a clinical isolate. The mutant strain displayed phenotypes typically associated with inactivation of morC. However, fimbriae were still observed on the surface, although in lesser amounts on some individual bacteria, and this strain formed a biofilm with volume similar to the parent. Interestingly, significant changes in microcolony architecture of the biofilm were observed by confocal microscopy. MorC plays a critical role in maintaining secretion of major virulence determinants of A. actinomycetemcomitans. Specific changes in the protein composition of the cell envelope indicate a direct or compensatory role of these proteins in maintaining membrane physiology. The functional conservation of MorC also implies an important role for this protein in other Gram-negative bacteria. This work suggests a role of MorC as an accessory or a scaffold protein involved in secretion

Structure-Based Genome Scale Function Prediction and Reconstruction of the Mycobacterium tuberculosis Metabolic Network

Due to vast improvements in sequencing methods over the past few decades, the availability of genomic data is rapidly increasing, thus bringing about the need for functional characterization tools. Considering the breadth of data involved, functional assays would be impractical and only a computational method could afford fast and cost-effective functional annotations. Therefore, homology-based computational methods are routinely used to assign putative molecular functions that can later be confirmed with targeted experiments. These methods are particularly well suited to predict the function of enzymes because most metabolic pathways are conserved across organisms. However, the current methods have limitations, especially when considering enzymes that have very low sequence and structure homology to well-annotated enzymes. We hypothesized that two enzymes with the same molecular function shared significant sequence homology in the region surrounding the active site, even if they appear diverged at the global sequence level. First, we have investigated the limits of sequence and structure conservation for enzymes with the same function during divergent evolution. The goal of this was to determine the sequence identity threshold beyond which functional annotations should not be transferred between two sequences; that is the level of homology beyond which the pair of proteins would not be expected to have the same function. Our analysis, which compares several models of sequence evolution, shows that the sequences of orthologous proteins catalyzing the same reaction rarely diverge beyond 30 % identity, even after approximately 3.5 billion years of evolution. As for structure conservation, enzymes catalyzing the same reactions rarely diverge beyond 3 Ã… root-mean-square distance (RMSD). We have also explored sequence conservation constraints as a function of the distance to the active site. Although residues closer to the protein active site (within a radius of 10 Ã… around the catalytic residues) are mutating significantly slower, the requirement to preserve the molecular function also constrains residues at other parts of the protein. From these results, we have developed a structure-based function prediction method where we employ active site conservation in addition to global sequence homology for functional characterization. We then integrated this method with a probabilistic whole-genome function prediction framework previously developed in the Vitkup group, GLOBUS. The original version of GLOBUS uses sampling of probability space to assign functions to all putative metabolic genes in an input genome by considering sequence homology to known enzymes, gene-gene context and EC co-occurrence. Applying this novel method to the whole-genome metabolic reconstruction of Mycobacterium tuberculosis, we made several novel predictions for genes with apparent links to pathogenesis. Notably, our predictions allowed us to reconstruct the cholesterol degradation pathway in M. tuberculosis, which has been implicated in bacterial persistence in the literature but remains to be fully characterized. This pathway is absent from previously published metabolic models of M. tuberculosis. Our new model can now be used to simulate different environments and conditions in order to gain a better understanding of the metabolic adaptability of M. tuberculosis during pathogenesis

Síntese enzímica de mono e dinucleosídeos polifosfatados : Acção catalítica da sintétase de Acil-Coenzima A de Pseudomonas fragi e da Luciférase de Photinus pyralis

Dissertação de Doutoramento em Medicina apresentada à Faculdade de Medicina da Universidade do Porto1 RESUMO E CONCLUSÕES A crescente convicção de que os mono e os dinucleosídeos polifosfatados são modu-ladores intra e extracelulares de processos biológicos justifica o nosso interesse pela sua síntese.Tendo sido formulada a hipótese de que a síntese dos dinucleosídeos polifosfatados poderia ser catalisada por enzimas capazes de formar acil-adenilatos com libertação concomitante de pirofosfato, estudámos esta possibilidade no caso concreto da sintétase de acil-CoA de Pseudomonas fragi (EC 6.2.1.3). Demonstrámos que esta enzima catalisa a síntese de adenosina-5 -tetrafosfato (p4A) e adenosina-5 -pentafosfato (p5A) a partir de ATP e tripolifosfato (P3) ou tetrapolifosfato (P4), respectivamente. O dATP, a adenosina-5 -O-[g-tiotrifosfato] (ATPgS), a adenosina(5 )tetrafosfo(5 )adenosina (Ap4A) e a adeno-sina(5 )pentafosfo(5 )adenosina (Ap5A) também eram substratos da reacção, gerando, na presença de P3, p4dA ou p4A. O UTP, o CTP ou o AMP não eram dadores de nucleotídeo. Os valores de Km para o ATP e o P3 eram, nas condições de ensaio utilizadas, 0,015 e 1,3 mM, respectivamente. A actividade catalítica dependia da presença de catiões bivalentes e um máximo de velocidade foi obtida na presença de MgCl2 ou CoCl2 equimolecular com o somatório de ATP e P3. As velocidades relativas de síntese de p4A com distintos catiões bivalentes foram: Mg=Co>Mn=Zn>>Ca. Um máximo e um mínimo de actividade foram medidos nos valores de pH de 5,5 e 8,2, respectivamente; o oposto foi observado no caso da actividade de síntese de palmitil-CoA, com um máximo a pH 8,2. A razão entre as actividades de síntese de palmitil-CoA e de p4A era cerca de 10 (a pH 5,5) e cerca de 200 (a pH 8,2). A coenzima A inibia a síntese de p4A e o seu efeito inibitório era antagonizado por ácidos gordos.Com uma actividade muito mais baixa, a sintétase de acil-CoA de Pseudomonas fragi também catalisava a síntese de Ap4A (a partir de ATP), Ap5A (a partir de p4A) e adenosina(5 )tetrafosfo(5 )nucleosídeos (Ap4N) a parti ..

The ENZYME data bank in 1999.

The ENZYME data bank is a repository of information related to the nomenclature of enzymes. In recent years it has become an indispensable resource for the development of metabolic databases. The current version contains information on 3704 enzymes. It is available through the ExPASy WWW server (http://www.expasy.ch/)