PubMed Health⌕ Search

Biomedical subjects

P M Coutinho

Publications and source records attributed to P M Coutinho.

At least 19 recordsLinked to original sources
PubMed2006

The genome of black cottonwood, Populus trichocarpa (Torr. & Gray).

We report the draft genome of the black cottonwood tree, Populus trichocarpa. Integration of shotgun sequence assembly with genetic mapping enabled chromosome-scale reconstruction of the genome. More than 45,000 putative protein-coding genes were identified. Analysis of the assembled genome revealed a whole-genome duplication event; about 8000 pairs of duplicated genes from that event survived in the Populus genome. A second, older duplication event is indistinguishably coincident with the divergence of the Populus and Arabidopsis lineages. Nucleotide substitution, tandem gene duplication, and gross chromosomal rearrangement appear to proceed substantially more slowly in Populus than in Arabidopsis. Populus has more protein-coding genes than Arabidopsis, ranging on average from 1.4 to 1.6 putative Populus homologs for each Arabidopsis gene. However, the relative frequency of protein domains in the two genomes is similar. Overrepresented exceptions in Populus include genes associated with lignocellulosic wall biosynthesis, meristem development, disease resistance, and metabolite transport.

Arabidopsis↗
PubMed2003

Cloning and sequence analysis of the ces10 gene encoding a Sphingomonas paucimobilis esterase.

The ces10 gene of the gellan gum-producing strain Sphingomonas paucimobilis ATCC 31461 was cloned and sequenced. Multi-sequence alignment of the deduced protein indicated that Ces10 belongs to the serine hydrolase family with a potential catalytic triad comprising Ser(153) (within the G-X-S-X-G consensus sequence), His(75) and Asp(125). The mixed block results obtained following pattern search and the low identities detected in a BLAST analysis indicate that Ces10 is significantly different from other characterised bacterial esterases/lipases. Nevertheless, the Ces10 amino acid sequence showed 45% similarity with Rhodococcus sp. heroin esterase and 48% with Bacillus subtilis p-nitrobenzyl esterase. Ces10, with a predicted molecular mass of 30,641 Da, was overproduced in Escherichia coli and purified to homogeneity in a histidine-tagged form. Enzyme assays using p-nitrophenyl-esters (p-NP-esters) with different acyl chain-lengths as the substrate confirmed the anticipated esterase activity. Ces10 exhibited a marked preference for short-chain fatty acids, yielding the highest activity with p-NP-propionate (optimal pH 7.4, optimal temperature 37 degrees C).

Amino Acid Sequence↗
PubMed2001

A census of carbohydrate-active enzymes in the genome of Arabidopsis thaliana.

The synthesis, modification, and breakdown of carbohydrates is one of the most fundamentally important reactions in nature. The structural and functional diversity of glycosides is mirrored by a vast array of enzymes involved in their synthesis (glycosyltransferases), modification (carbohydrate esterases) and breakdown (glycoside hydrolases and polysaccharide lyases). The importance of these processes is reflected in the dedication of 1-2% of an organism's genes to glycoside hydrolases and glycosyltransferases alone. In plants, these processes are of particular importance for cell-wall synthesis and expansion. starch metabolism, defence against pathogens, symbiosis and signalling. Here we present an analysis of over 730 open reading frames representing the two main classes of carbohydrate-active enzymes, glycoside hydrolases and glycosyltransferases, in the genome of Arabidopsis thaliana. The vast importance of these enzymes in cell-wall formation and degradation is revealed along with the unexpected dominance of pectin degradation in Arabidopsis, with at least 170 open-reading frames dedicated solely to this task.

Arabidopsis↗
PubMed2000

Replacement and deletion mutations in the catalytic domain and belt region of Aspergillus awamori glucoamylase to enhance thermostability.

Three single-residue mutations, Asp71-->Asn, Gln409-->Pro and Gly447-->Ser, two long-to-short loop replacement mutations, Gly23-Ala24-Asp25-Gly26-Ala27-Trp28- Val29-Ser30-->Asn-Pro-Pro (23-30 replacement) and Asp297-Ser298-Glu299-Ala300-Val301-->Ala-G ly-Ala (297-301 replacement) and one deletion mutation removing Glu439, Thr440 and Ser441 (Delta439-441), all based on amino acid sequence alignments, were made to improve Aspergillus awamori glucoamylase thermostability. The first and second single-residue mutations were designed to introduce a potential N:-glycosylation site and to restrict backbone bond rotation, respectively, and therefore to decrease entropy during protein unfolding. The third single-residue mutation was made to decrease flexibility and increase O:-glycosylation in the already highly O:-glycosylated belt region that extends around the globular catalytic domain. The 23-30 replacement mutation was designed to eliminate a very thermolabile extended loop on the catalytic domain surface and to bring the remainder of this region closer to the rest of the catalytic domain, therefore preventing it from unfolding. The 297-301 replacement mutant GA was made to understand the function of the random coil region between alpha-helices 9 and 10. Delta439-441 was constructed to decrease belt flexibility. All six mutations increased glucoamylase thermostability without significantly changing enzyme kinetic properties, with the 23-30 replacement mutation increasing the activation free energy for thermoinactivation by about 4 kJ/mol, which leads to a 4 degrees C increase in operating temperature at constant thermostability.

Aspergillus↗
PubMed1999

Cellulosome-like sequences in Archaeoglobus fulgidus: an enigmatic vestige of cohesin and dockerin domains.

The distribution of cellulosomal cohesin domains among the sequences currently compiled in various sequence databases was investigated. Two cohesin domains were detected in two consecutive open reading frames (ORFs) of the recently sequenced genome of the archaeon Archaeoglobus fulgidus. Otherwise, no cohesin-like sequence could be detected in organisms other than those of the Eubacteria. One of the A. fulgidus cohesin-containing ORFs also harbored a dockerin domain, but the additional modular portions of both genes are undefined, both with respect to sequence homology and function. It is currently unclear what function(s) the putative cohesin and dockerin-containing proteins play in the life cycle of this organism. In particular, since A. fulgidus contains no known glycosyl hydrolase gene, the presence of a cellulosome can be excluded. The results suggest that cohesin and dockerin signature sequences cannot be used alone for the definitive identification of cellulosomes in genomes.

Amino Acid Sequence↗
PubMed1999

Automated docking of maltose, 2-deoxymaltose, and maltotetraose into the soybean beta-amylase active site.

In this study, products and substrates were docked into the active site of beta-amylase using the simulated annealing algorithm AutoDock. Lowest-energy conformers reproduced known crystallographic atom positions within 0.4 to 0.8 A rmsd. Docking studies were carried out with both open and closed configurations of the beta-amylase mobile flap, a loop comprising residues 96 to 103. Ligands with two rings docked within the cleft near the active site when the flap was open, but those with four rings did not. The flap must be closed for alpha-maltotetraose to adopt a conformation allowing it to dock near the crystallographically determined subsites. The closed flap is necessary for productive but not for nonproductive binding, and therefore it plays a essential role in catalysis. The gain in total binding energy upon closing of the flap for alpha-maltose docked to subsites -2, -1 and +1, +2 is about 22 kcal/mol, indicating more favorable interactions are possible with the flap closed. Larger intermolecular interaction energies are observed for two alpha-maltose molecules docked to subsites -2, -1 and +1, +2 than for one alpha-maltotetraose molecule docked from subsites -2 to +2, suggesting that it is only upon cleavage of the alpha-1,4 linkage that optimal closed-flap binding can occur with the crytallographically determined enzyme structure.

Binding Sites↗
PubMed1999

Life with no sugars?

Explore the source record for details and available documents.

Archaeoglobus fulgidus↗
PubMed1998

Mutations to alter Aspergillus awamori glucoamylase selectivity. I. Tyr48Phe49-->Trp, Tyr116-->Trp, Tyr175-->Phe, Arg241-->Lys, Ser411-->Ala and Ser411-->Gly.

Glucoamylase mutations to reduce isomaltose formation from glucose condensation and thus increase glucose yield from starch hydrolysis were designed to produce minor changes in the active site at positions not totally conserved. Tyr175-->Phe and Ser411-->Gly glucoamylases had catalytic efficiencies on DP 2-7 maltooligosaccharides like those of wild-type glucoamylase, while the catalytic efficiencies of Tyr116-->Trp, Arg241-->Lys and Ser411-->Ala glucoamylases were reduced by about half and Tyr48Phe49-->Trp glucoamylase had little remaining activity. Tyr175-->Phe, Ser411-->Ala and Ser411-->Gly glucoamylases had decreased ratios of the initial rate of isomaltose formation from glucose condensation to that of glucose formation from maltodextrin hydrolysis at both 35 and 55 degrees C compared with wild-type glucoamylase. Arg241-->Lys glucoamylase had a very similar ratio, while Tyr116-->Trp glucoamylase had a higher ratio. The highest glucose yields from maltodextrin hydrolysis were by the mutant glucoamylases having the lowest ratios of initial rates of isomaltose formation to glucose formation and this predicted high glucose yields better than the ratio of catalytic efficiency for maltose hydrolysis to that for isomaltose hydrolysis.

Arginine↗
PubMed1998

Mutations to alter Aspergillus awamori glucoamylase selectivity. III. Asn20-->Cys/Ala27-->Cys, Ala27-->Pro, Ser30-->Pro, Lys108-->Arg, Lys108-->Met, Gly137-->Ala, 311-314 Loop, Tyr312-->Trp and Ser436-->Pro.

Mutations Asn20-->Cys/Ala27-->Cys (SS), Ala27-->Pro, Ser30-->Pro, Lys108-->Arg, Gly137-->Ala, Tyr312-->Trp and Ser436-->Pro in Aspergillus awamori glucoamylase, along with a mutation inserting a seven-residue loop between Tyr311 and Gly314 (311-314 Loop), were made to increase glucose yield from maltodextrin hydrolysis. No active Lys108-->Met glucoamylase was found in the supernatant after being expressed from yeast. Lys108-->Arg, 311-314 Loop and Tyr312-->Trp glucoamylases have lower activities than wild-type glucoamylase; other GAs have the same or higher activities. SS and 311-314 Loop glucoamylases give one-quarter to two-thirds the relative rates of isomaltose formation from glucose compared with glucose formation from maltodextrins at 35, 45 and 55 degrees C, correlating with up to 2% higher peak glucose yields from 30% (w/v) maltodextrin hydrolysis. Conversely, Lys108-->Arg glucoamylase has relative isomaltose formation rates three times higher and glucose yields up to 4% lower than wild-type glucoamylase. Gly137-->Ala and Tyr312-->Trp glucoamylases also give high glucose yields at higher temperatures. Mutated glucoamylases that catalyze high rates of isomaltose formation give higher glucose yields from shorter than from longer maltodextrins, opposite to normal experience with more efficient glucoamylases.

Aspergillus↗
PubMed1998

Effect on thermostability and catalytic activity of introducing disulfide bonds into Aspergillus awamori glucoamylase.

Two additional disulfide bonds and three combined thermostabilizing mutations were introduced into Aspergillus awamori glucoamylase to test their effects on enzyme thermostability and catalytic properties. The single cysteine mutations N20C, A27C, T72C and A471C were made and combined to produce the double cysteine mutations N20C/ A27C and T72C/A471C. The double cysteine mutants were expressed efficiently in Saccharomyces cerevisiae, and disulfide bonds formed spontaneously after fermentation. At 50 degrees C, the single mutants N20C and A27C had decreased specific activity, whereas the specific activity of the double mutants N20C/A27C and T72C/A471C were similar to wild-type glucoamylase. The N20C/A27C mutation increased thermostability, with an increased activation free energy of 1.5 kJ/mol at 65 degrees C, while the single mutation A27C only slightly increased thermostability and N20C decreased it. The other disulfide bond-forming mutation T72C/A471C did not affect thermostability at pH 4.5. The N20C/A27C mutation was separately combined with two other thermostabilizing mutations, G137A and S436P. Thermostabilities of all of the combined mutated glucoamylases were additive. N20C/A27C/G137A glucoamylase had higher specific activity than wild-type glucoamylase from 45 to 67.5 degrees C. The disulfide bond between positions 20 and 27 connects the C-terminus of helix 1 and the following beta-turn, suggesting that this region is important for glucoamylase thermostability.

Aspergillus↗
PubMed1998

Stabilization of Aspergillus awamori glucoamylase by proline substitution and combining stabilizing mutations.

To stabilize Aspergillus awamori glucoamylase (GA), three proline substitution mutations were constructed. When expressed in Saccharomyces cerevisiae, Ser30-->Pro (S30P) stabilized the enzyme without decreased activity, whereas Asp345-->Pro (D345P) did not significantly alter and Glu408-->Pro (E408P) greatly decreased enzyme thermostability. The S30P mutation was combined with two previously identified stabilizing mutations: Gly137-->Ala, and Asn20-->Cys/Ala27-->Cys (which creates a disulfide bond between positions 20 and 27). The combined mutants demonstrated cumulative stabilization as shown by decreased irreversible thermoinactivation rates between 65 and 80 degrees C. Additionally, two of the combined mutants outperformed wild-type GA in high-temperature (65 degrees C) saccharifications of DE 10 maltodextrin and were more active than the wild-type enzyme when assayed using maltose as substrate.

Amino Acid Substitution↗
PubMed1997

Automated docking of monosaccharide substrates and analogues and methyl alpha-acarviosinide in the glucoamylase active site.

Glucoamylase is an important industrial glucohydrolase with a large specificity range. To investigate its interaction with the monosaccharides D-glucose, D-mannose, and D-galactose and with the substrate analogues 1-deoxynojirimycin, D-glucono-1,5-lactone, and methyl alpha-acarviosinide, MM3(92)-optimized structures were docked into its active site using AutoDock 2.1. The results were compared to structures of glucoamylase complexes obtained by protein crystallography. Charged forms of some substrate analogues were also docked to assess the degree of protonation possessed by glucoamylase inhibitors. Many forms of methyl alpha-acarviosinide were conformationally mapped by using MM3(92), characterizing the conformational pH dependence found for the acarbose family of glucosidase inhibitors. Their significant conformers, representing the most common states of the inhibitor, were used as initial structures for docking. This constitutes a new approach for the exploration of binding modes of carbohydrate chains. Docking results differ slightly from x-ray crystallographic data, the difference being of the order of the crystallographic error. The estimated energetic interactions, even though agreeing in some cases with experimental binding kinetics, are only qualitative due to the large approximations made by AutoDock force field.

1-Deoxynojirimycin↗
PubMed1997

Glucoamylase structural, functional, and evolutionary relationships.

To correlate structural features with glucoamylase properties, a structure-based multisequence alignment was constructed using information from catalytic and starch-binding domain models. The catalytic domain is composed of three hydrophobic folding units, the most labile and least hydrophobic of them being missing in the most stable glucoamylase. The role of O-glycosylation in stabilizing the most hydrophobic folding unit, the only one where thermostabilizing mutations with unchanged activity have been made, is described. Differences in both length and composition of interhelical loops are correlated with stability and selectivity characteristics. Two new glucoamylase subfamilies are defined by using homology criteria. Protein parsimony analysis suggests an ancient bacterial origin for the glucoamylase gene. Increases in length of the belt surrounding the active site, degree of O-glycosylation, and length of the linker probably correspond to evolutionary steps that increase stability and secretion levels of Aspergillus-related glucoamylases.

Amino Acid Sequence↗
PubMed1997

Automated docking of glucosyl disaccharides in the glucoamylase active site.

To better understand the molecular basis of glucomylase selectivity, low-energy conformers of glucosyl disaccharides obtained from relaxed-residue conformational mapping were flexibly docked into the glucoamylase active site using AutoDock 2.2. This procedure ensures that significant conformational space is searched and can produce bound structures comparable to those obtained by protein crystallography. alpha-Linked glucosyl disaccharides except alpha,alpha-trehalose dock easily into the active site while exclusively beta-linked disaccharides do not, explaining why only the former are glucoamylase substrates. The optimized docking modes are similar at the nonreducing end of the different substrates. Individual atomic energies of intermolecular interaction allow the definite identification of key hydroxyl groups for each substrate. This approach confirmed the versatility of the second subsite of the glucoamylase active site in binding different substrates.

Binding Sites↗
PubMed1995

Deletion analysis of the starch-binding domain of Aspergillus glucoamylase.

The large form of glucoamylase (GAI) from Aspergillus awamori (EC 3.2.1.3) binds strongly to native granular starch, whereas a truncated form (GAII) which lacks 103 C-terminal residues, does not. This C-terminal region, conserved among fungal glucoamylases and other starch-degrading enzymes, is part of an independent starch-binding domain (SBD). To investigate the SBD boundaries and the function of conserved residues in two putative substrate-binding sites, five gluco-amylase mutants were constructed with extensive deletions in this region for expression in Saccharomyces cerevisiae. Progressive loss of both starch-binding and starch-hydrolytic activity occurred upon removal of eight and 25 C-terminal amino acid residues, or 21 and 52 residues close to the N-terminus, confirming the requirement for the entire region in formation of a functional SBD. C-terminal deletions strongly impaired SBD function, suggesting a more important role for one of the putative binding sites. A GAII phenocopy showed a nearly complete loss of starch-binding and starch-hydrolytic activity. The deletions did not affect enzyme activity on soluble starch or thermo-stability of the enzyme, confirming the independence of the catalytic domain from the SBD.

Amino Acid Sequence↗
PubMed1994

Reading-frame shift in Saccharomyces glucoamylases restores catalytic base, extends sequence and improves alignment with other glucoamylases.

A recent article [Coutinho and Reilly (1994) Protein Engng, 7, 749-760] presented the alignment of 14 glucoamylases by hydrophobic cluster analysis. The catalytic bases of two of these glucoamylases, from Saccharomyces cerevisiae and Saccharomyces diastaticus, were not conserved, opening the possibility of a reading-frame shift error in a segment coding for amino acids near the apparent C-termini of the mature proteins. Indeed, an addition of one nucleotide restores the catalytic base, extends the sequence by 39 residues and greatly improves the amino acid alignment in this region.

Amino Acid Sequence↗