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1.
Nature ; 452(7183): 88-92, 2008 Mar 06.
Article in English | MEDLINE | ID: mdl-18322534

ABSTRACT

Mycorrhizal symbioses--the union of roots and soil fungi--are universal in terrestrial ecosystems and may have been fundamental to land colonization by plants. Boreal, temperate and montane forests all depend on ectomycorrhizae. Identification of the primary factors that regulate symbiotic development and metabolic activity will therefore open the door to understanding the role of ectomycorrhizae in plant development and physiology, allowing the full ecological significance of this symbiosis to be explored. Here we report the genome sequence of the ectomycorrhizal basidiomycete Laccaria bicolor (Fig. 1) and highlight gene sets involved in rhizosphere colonization and symbiosis. This 65-megabase genome assembly contains approximately 20,000 predicted protein-encoding genes and a very large number of transposons and repeated sequences. We detected unexpected genomic features, most notably a battery of effector-type small secreted proteins (SSPs) with unknown function, several of which are only expressed in symbiotic tissues. The most highly expressed SSP accumulates in the proliferating hyphae colonizing the host root. The ectomycorrhizae-specific SSPs probably have a decisive role in the establishment of the symbiosis. The unexpected observation that the genome of L. bicolor lacks carbohydrate-active enzymes involved in degradation of plant cell walls, but maintains the ability to degrade non-plant cell wall polysaccharides, reveals the dual saprotrophic and biotrophic lifestyle of the mycorrhizal fungus that enables it to grow within both soil and living plant roots. The predicted gene inventory of the L. bicolor genome, therefore, points to previously unknown mechanisms of symbiosis operating in biotrophic mycorrhizal fungi. The availability of this genome provides an unparalleled opportunity to develop a deeper understanding of the processes by which symbionts interact with plants within their ecosystem to perform vital functions in the carbon and nitrogen cycles that are fundamental to sustainable plant productivity.


Subject(s)
Basidiomycota/genetics , Basidiomycota/physiology , Genome, Fungal/genetics , Mycorrhizae/genetics , Mycorrhizae/physiology , Plant Roots/microbiology , Symbiosis/physiology , Abies/microbiology , Abies/physiology , Basidiomycota/enzymology , Fungal Proteins/classification , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gene Expression Regulation , Genes, Fungal/genetics , Hyphae/genetics , Hyphae/metabolism , Mycorrhizae/enzymology , Plant Roots/physiology , Symbiosis/genetics
2.
Science ; 313(5793): 1596-604, 2006 Sep 15.
Article in English | MEDLINE | ID: mdl-16973872

ABSTRACT

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.


Subject(s)
Gene Duplication , Genome, Plant , Populus/genetics , Sequence Analysis, DNA , Arabidopsis/genetics , Chromosome Mapping , Computational Biology , Evolution, Molecular , Expressed Sequence Tags , Gene Expression , Genes, Plant , Oligonucleotide Array Sequence Analysis , Phylogeny , Plant Proteins/chemistry , Plant Proteins/genetics , Polymorphism, Single Nucleotide , Populus/growth & development , Populus/metabolism , Protein Structure, Tertiary , RNA, Plant/analysis , RNA, Untranslated/analysis
3.
Appl Microbiol Biotechnol ; 61(5-6): 517-22, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12764567

ABSTRACT

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).


Subject(s)
Esterases/genetics , Genes, Bacterial , Sphingomonas/enzymology , Sphingomonas/genetics , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Base Sequence , Cloning, Molecular , DNA, Bacterial/genetics , Esterases/chemistry , Esterases/metabolism , Molecular Sequence Data , Molecular Weight , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Substrate Specificity
4.
Plant Mol Biol ; 47(1-2): 55-72, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11554480

ABSTRACT

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.


Subject(s)
Arabidopsis/enzymology , Carbohydrate Metabolism , Enzymes/metabolism , Genome, Plant , Arabidopsis/genetics , Arabidopsis/metabolism , Cell Wall/metabolism , Enzymes/chemistry , Enzymes/genetics , Glycoside Hydrolases/chemistry , Glycoside Hydrolases/genetics , Glycoside Hydrolases/metabolism , Glycosyltransferases/chemistry , Glycosyltransferases/genetics , Glycosyltransferases/metabolism , Open Reading Frames , Protein Conformation
6.
Protein Eng ; 13(9): 655-9, 2000 Sep.
Article in English | MEDLINE | ID: mdl-11054460

ABSTRACT

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.


Subject(s)
Aspergillus/enzymology , Glucan 1,4-alpha-Glucosidase/chemistry , Glucan 1,4-alpha-Glucosidase/genetics , Mutation , Base Sequence , Catalytic Domain , Enzyme Stability , Glucan 1,4-alpha-Glucosidase/metabolism , Glycosylation , Kinetics , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Conformation , Protein Engineering/methods , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Deletion , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
7.
FEBS Lett ; 463(3): 277-80, 1999 Dec 17.
Article in English | MEDLINE | ID: mdl-10606737

ABSTRACT

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.


Subject(s)
Archaeoglobus fulgidus/chemistry , Indoles/chemistry , Nuclear Proteins/chemistry , Propionates/chemistry , Amino Acid Sequence , Archaeoglobus fulgidus/genetics , Cell Cycle Proteins , Chromosomal Proteins, Non-Histone , Fungal Proteins , Molecular Sequence Data , Open Reading Frames , Phylogeny , Sequence Alignment , Cohesins
8.
Proteins ; 37(2): 166-75, 1999 Nov 01.
Article in English | MEDLINE | ID: mdl-10584063

ABSTRACT

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.


Subject(s)
Glycine max/chemistry , Maltose/analogs & derivatives , Maltose/chemistry , beta-Amylase/chemistry , Binding Sites , Carbohydrate Sequence , Models, Molecular , Molecular Sequence Data , Oligosaccharides/chemistry , Protein Conformation
10.
Protein Eng ; 11(9): 783-8, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9796827

ABSTRACT

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.


Subject(s)
Aspergillus/enzymology , Glucan 1,4-alpha-Glucosidase/chemistry , Proline/chemistry , Amino Acid Substitution , Base Sequence , Carbohydrate Metabolism , DNA Primers , Glucan 1,4-alpha-Glucosidase/antagonists & inhibitors , Glucan 1,4-alpha-Glucosidase/genetics , Glucan 1,4-alpha-Glucosidase/metabolism , Hot Temperature , Kinetics , Models, Molecular , Mutagenesis , Protein Conformation
11.
Protein Eng ; 11(8): 661-7, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9749918

ABSTRACT

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.


Subject(s)
Aspergillus/enzymology , Glucan 1,4-alpha-Glucosidase/genetics , Glucan 1,4-alpha-Glucosidase/metabolism , Disulfides , Enzyme Activation , Enzyme Stability/genetics , Glucan 1,4-alpha-Glucosidase/chemistry , Hot Temperature , Models, Molecular , Mutation , Protein Conformation , Protein Engineering/methods , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
12.
Protein Eng ; 11(5): 389-98, 1998 May.
Article in English | MEDLINE | ID: mdl-9681872

ABSTRACT

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.


Subject(s)
Aspergillus/enzymology , Glucan 1,4-alpha-Glucosidase/genetics , Glucan 1,4-alpha-Glucosidase/metabolism , Mutagenesis, Site-Directed , Glucan 1,4-alpha-Glucosidase/chemistry , Glucose/metabolism , Hydrolysis , Isomaltose/metabolism , Kinetics , Maltose/metabolism , Models, Molecular , Polysaccharides/metabolism , Protein Denaturation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism
13.
Protein Eng ; 11(2): 119-26, 1998 Feb.
Article in English | MEDLINE | ID: mdl-9605546

ABSTRACT

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.


Subject(s)
Aspergillus/enzymology , Glucan 1,4-alpha-Glucosidase/genetics , Glucan 1,4-alpha-Glucosidase/metabolism , Mutagenesis, Site-Directed , Arginine , Binding Sites , Carbohydrate Conformation , Catalysis , Glucan 1,4-alpha-Glucosidase/chemistry , Glucose/metabolism , Hydrolysis , Isomaltose/metabolism , Kinetics , Molecular Structure , Polysaccharides/metabolism , Serine , Structure-Activity Relationship , Thermodynamics , Tyrosine
14.
Proteins ; 29(3): 334-47, 1997 Nov.
Article in English | MEDLINE | ID: mdl-9365988

ABSTRACT

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.


Subject(s)
Aspergillus niger/enzymology , Evolution, Molecular , Glucan 1,4-alpha-Glucosidase/chemistry , Amino Acid Sequence , Aspergillus , Binding Sites , Catalysis , Glycosylation , Molecular Sequence Data , Protein Conformation , Protein Folding , Sequence Alignment
15.
Proteins ; 28(2): 162-73, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9188734

ABSTRACT

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.


Subject(s)
Disaccharides/metabolism , Glucan 1,4-alpha-Glucosidase/metabolism , Binding Sites , Carbohydrate Conformation , Catalysis , Hydrogen Bonding
16.
Proteins ; 27(2): 235-48, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9061788

ABSTRACT

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.


Subject(s)
1-Deoxynojirimycin/chemistry , Computer Simulation , Disaccharides/chemistry , Galactose/chemistry , Glucan 1,4-alpha-Glucosidase/chemistry , Glucan 1,4-alpha-Glucosidase/metabolism , Gluconates/chemistry , Glucose/chemistry , Mannose/chemistry , Models, Molecular , 1-Deoxynojirimycin/metabolism , Aspergillus/enzymology , Automation , Binding Sites , Catalysis , Disaccharides/metabolism , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Galactose/metabolism , Gluconates/metabolism , Glucose/metabolism , Lactones , Mannose/metabolism , Monosaccharides/chemistry , Monosaccharides/metabolism , Substrate Specificity
18.
Protein Eng ; 8(10): 1049-55, 1995 Oct.
Article in English | MEDLINE | ID: mdl-8771186

ABSTRACT

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.


Subject(s)
Aspergillus/enzymology , Aspergillus/genetics , Glucan 1,4-alpha-Glucosidase/chemistry , Glucan 1,4-alpha-Glucosidase/genetics , Amino Acid Sequence , Base Sequence , Binding Sites/genetics , Conserved Sequence , DNA, Fungal/genetics , Enzyme Stability/genetics , Glucan 1,4-alpha-Glucosidase/metabolism , Molecular Sequence Data , Molecular Structure , Plasmids/genetics , Protein Engineering , Saccharomyces cerevisiae/genetics , Sequence Deletion , Starch , Temperature
19.
Protein Eng ; 7(11): 1281-2, 1994 Nov.
Article in English | MEDLINE | ID: mdl-7700859

ABSTRACT

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.


Subject(s)
Glucan 1,4-alpha-Glucosidase/genetics , Reading Frames/genetics , Saccharomyces/genetics , Amino Acid Sequence , Artifacts , Base Sequence , Conserved Sequence , Databases, Factual , Molecular Sequence Data , Saccharomyces/enzymology , Sequence Homology, Amino Acid
20.
Protein Eng ; 7(8): 1005-12, 1994 Aug.
Article in English | MEDLINE | ID: mdl-7809026

ABSTRACT

Seven thermosensitive glucoamylase mutants generated by random mutagenesis and expressed in Saccharomyces cerevisiae were sequenced and their inactivation kinetics were determined. Wild-type glucoamylase expressed in S. cerevisiae was more glycosylated and more stable than the native Aspergillus niger enzyme. All mutants had lower free energies of inactivation than wild-type glucoamylase. In the Ala39-->Val, Ala302-->Val and Leu410-->Phe mutants, small hydrophobic residues were replaced by larger ones, showing that increases in size and hydrophobicity of residues included in hydrophobic clusters were destabilizing. The Gly396-->Ser and Gly407-->Asp mutants had very flexible residues replaced by more rigid ones, and this probably induced changes in the backbone conformation that destabilized the protein. The Pro128-->Ser mutation changed a rigid residue in an alpha-helix to a more flexible one, and destabilized the protein by increasing the entropy of the unfolded state. The Ala residue in the Ala442-->Thr mutation is in the highly O-glycosylated region surrounded by hydrophilic residues, where it may be a hydrophobic anchor linking the O-glycosylated arm to the catalytic core. It was replaced by a residue that potentially is O-glycosylated. In five of the seven mutations, residues that were part of hydrophobic microdomains were changed, confirming the importance of the latter in protein stability and structure.


Subject(s)
Aspergillus/enzymology , Glucan 1,4-alpha-Glucosidase/chemistry , Amino Acid Sequence , Aspergillus/genetics , Enzyme Stability , Glucan 1,4-alpha-Glucosidase/genetics , Hot Temperature , Models, Molecular , Molecular Sequence Data , Mutagenesis , Protein Conformation , Protein Folding , Protein Structure, Secondary , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Sequence Analysis, DNA , Structure-Activity Relationship , Thermodynamics
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