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1.
Acta Crystallogr F Struct Biol Commun ; 71(Pt 1): 66-70, 2015 Jan 01.
Article in English | MEDLINE | ID: mdl-25615972

ABSTRACT

The enzyme-catalysed degradation of starch is central to many industrial processes, including sugar manufacture and first-generation biofuels. Classical biotechnological platforms involve steam explosion of starch followed by the action of endo-acting glycoside hydrolases termed α-amylases and then exo-acting α-glucosidases (glucoamylases) to yield glucose, which is subsequently processed. A key enzymatic player in this pipeline is the `Termamyl' class of bacterial α-amylases and designed/evolved variants thereof. Here, the three-dimensional structure of one such Termamyl α-amylase variant based upon the parent Geobacillus stearothermophilus α-amylase is presented. The structure has been solved at 1.9 Šresolution, revealing the classical three-domain fold stabilized by Ca2+ and a Ca2+-Na+-Ca2+ triad. As expected, the structure is similar to the G. stearothermophilus α-amylase but with main-chain deviations of up to 3 Šin some regions, reflecting both the mutations and differing crystal-packing environments.


Subject(s)
Bacterial Proteins/chemistry , Geobacillus stearothermophilus/enzymology , alpha-Amylases/chemistry , Amino Acid Sequence , Catalytic Domain , Crystallography, X-Ray , Hydrogen Bonding , Models, Molecular , Protein Structure, Secondary , Structural Homology, Protein
2.
Biochemistry ; 51(26): 5339-47, 2012 Jul 03.
Article in English | MEDLINE | ID: mdl-22668005

ABSTRACT

The stability of serine proteases is of major importance for their application in industrial processes. Here we study the determinants of the stability of a Nocardiopsis prasina serine protease using fast residual activity assays, a feature classification algorithm, and structure-based energy calculation algorithms for 121 micropurified mutant enzyme clones containing multiple point mutations. Using a multivariate regression analysis, we deconvolute the data for the mutant clones and find that mutations of residues Asn47 and Pro124 are deleterious to the stability of the enzyme. Both of these residues are situated in loops that are known to be important for the stability of the highly homologous α-lytic protease. Structure-based energy calculations with PEATSA give a good general agreement with the trend of experimentally measured values but also identify a number of clones that the algorithm fails to predict correctly. We discuss the significance of the results in relation to the structure and function of closely related proteases, comment on the optimal experimental design when performing high-throughput experiments for characterizing the determinants of protein stability, and discuss the performance of structure-based energy calculations with complex data sets such as the one presented here.


Subject(s)
Actinomycetales/enzymology , Serine Proteases/chemistry , Serine Proteases/metabolism , Calorimetry, Differential Scanning , Circular Dichroism , Mutation , Protein Stability , Serine Proteases/genetics , Structure-Activity Relationship
3.
Carbohydr Res ; 346(13): 2028-33, 2011 Sep 27.
Article in English | MEDLINE | ID: mdl-21696710

ABSTRACT

ß-1,4-Galactanases belong to glycoside hydrolase family GH 53 and degrade galactan and arabinogalactan side chains of the complex pectin network in plant cell walls. Two fungal ß-1,4-galactanases from Aspergillus aculeatus, Meripileus giganteus and one bacterial enzyme from Bacillus licheniformis have been kinetically characterized using the chromogenic substrate analog 4-nitrophenyl ß-1,4-d-thiogalactobioside synthesized by the thioglycoligase approach. Values of k(cat)/K(m) for this substrate with A. aculeatus ß-1,4-galactanase at pH 4.4 and for M. giganteus ß-1,4-galactanase at pH 5.5 are 333M(-1)s(-1) and 62M(-1)s(-1), respectively. By contrast the B. licheniformis ß-1,4-galactanase did not hydrolyze 4-nitrophenyl ß-1,4-d-thiogalactobioside. The different kinetic behavior observed between the two fungal and the bacterial ß-1,4-galactanases can be ascribed to an especially long loop 8 observed only in the structure of B. licheniformis ß-1,4-galactanase. This loop contains substrate binding subsites -3 and -4, which presumably cause B. licheniformis ß-1,4-galactanase to bind 4-nitrophenyl -1,4-ß-d-thiogalactobioside non-productively. In addition to their cleavage of 4-nitrophenyl -1,4-ß-d-thiogalactobioside, the two fungal enzymes also cleaved the commercially available 2-nitrophenyl-1,4-ß-d-galactopyranoside, but kinetic parameters could not be determined because of transglycosylation at substrate concentrations above 4mM.


Subject(s)
Chromogenic Compounds/chemistry , Chromogenic Compounds/metabolism , Glycoside Hydrolases/metabolism , Carbohydrate Sequence , Chromatography, Thin Layer , Galactose/chemistry , Galactose/metabolism , Molecular Sequence Data , Substrate Specificity
4.
J Mol Biol ; 404(1): 100-11, 2010 Nov 19.
Article in English | MEDLINE | ID: mdl-20851126

ABSTRACT

We present here the first experimental evidence for bound substrate in the active site of a rhamnogalacturonan lyase belonging to family 4 of polysaccharide lyases, Aspergillus aculeatus rhamnogalacturonan lyase (RGL4). RGL4 is involved in the degradation of rhamnogalacturonan-I, an important pectic plant cell wall polysaccharide. Based on the previously determined wild-type structure, enzyme variants RGL4_H210A and RGL4_K150A have been produced and characterized both kinetically and structurally, showing that His210 and Lys150 are key active-site residues. Crystals of the RGL4_K150A variant soaked with a rhamnogalacturonan digest gave a clear picture of substrate bound in the -3/+3 subsites. The crystallographic and kinetic studies on RGL4, and structural and sequence comparison to other enzymes in the same and other PL families, enable us to propose a detailed reaction mechanism for the ß-elimination on [-,2)-α-l-rhamno-(1,4)-α-d-galacturonic acid-(1,-]. The mechanism differs significantly from the one established for pectate lyases, in which most often calcium ions are engaged in catalysis.


Subject(s)
Aspergillus/enzymology , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Polysaccharide-Lyases/chemistry , Polysaccharide-Lyases/metabolism , Amino Acid Substitution , Catalytic Domain , Crystallography, X-Ray , Kinetics , Models, Molecular , Mutagenesis, Site-Directed , Mutant Proteins/chemistry , Mutant Proteins/genetics , Pectins/chemistry , Pectins/metabolism , Protein Binding , Protein Structure, Tertiary
5.
Biochemistry ; 48(29): 7009-18, 2009 Jul 28.
Article in English | MEDLINE | ID: mdl-19441796

ABSTRACT

The mechanism by which polysaccharide-hydrolyzing enzymes manifest specificity toward heterogeneous substrates, in which the sequence of sugars is variable, is unclear. An excellent example of such heterogeneity is provided by the plant structural polysaccharide glucomannan, which comprises a backbone of beta-1,4-linked glucose and mannose units. beta-Mannanases, located in glycoside hydrolase (GH) families 5 and 26, hydrolyze glucomannan by cleaving the glycosidic bond of mannosides at the -1 subsite. The mechanism by which these enzymes select for glucose or mannose at distal subsites, which is critical to defining their substrate specificity on heterogeneous polymers, is currently unclear. Here we report the biochemical properties and crystal structures of both a GH5 mannanase and a GH26 mannanase and describe the contributions to substrate specificity in these enzymes. The GH5 enzyme, BaMan5A, derived from Bacillus agaradhaerens, can accommodate glucose or mannose at both its -2 and +1 subsites, while the GH26 Bacillus subtilis mannanase, BsMan26A, displays tight specificity for mannose at its negative binding sites. The crystal structure of BaMan5A reveals that a polar residue at the -2 subsite can make productive contact with the substrate 2-OH group in either its axial (as in mannose) or its equatorial (as in glucose) configuration, while other distal subsites do not exploit the 2-OH group as a specificity determinant. Thus, BaMan5A is able to hydrolyze glucomannan in which the sequence of glucose and mannose is highly variable. The crystal structure of BsMan26A in light of previous studies on the Cellvibrio japonicus GH26 mannanases CjMan26A and CjMan26C reveals that the tighter mannose recognition at the -2 subsite is mediated by polar interactions with the axial 2-OH group of a (4)C(1) ground state mannoside. Mutagenesis studies showed that variants of CjMan26A, from which these polar residues had been removed, do not distinguish between Man and Glc at the -2 subsite, while one of these residues, Arg 361, confers the elevated activity displayed by the enzyme against mannooligosaccharides. The biological rationale for the variable recognition of Man- and Glc-configured sugars by beta-mannanases is discussed.


Subject(s)
beta-Mannosidase/metabolism , Bacillus/enzymology , Base Sequence , Crystallography , DNA Primers , Models, Molecular , Mutagenesis, Site-Directed , Polymerase Chain Reaction , Substrate Specificity , beta-Mannosidase/chemistry , beta-Mannosidase/genetics
7.
J Mol Biol ; 385(3): 924-37, 2009 Jan 23.
Article in English | MEDLINE | ID: mdl-18992756

ABSTRACT

Theoretical, computational, and experimental studies have suggested the existence of solvation barriers in protein unfolding and denaturation processes. These barriers are related to the finite size of water molecules and can be envisioned as arising from the asynchrony between water penetration and breakup of internal interactions. Solvation barriers have been proposed to play roles in protein cooperativity and kinetic stability; therefore, they may be expected to be subject to natural selection. We study the thermal denaturation, in the presence and in the absence of chemical denaturants, of triosephosphate isomerases (TIMs) from three different species: Trypanosoma cruzi, Trypanosoma brucei, and Leishmania mexicana. In all cases, denaturation was irreversible and kinetically controlled. Surprisingly, however, we found large differences between the kinetic denaturation parameters, with T. cruzi TIM showing a much larger activation energy value (and, consequently, much lower room-temperature, extrapolated denaturation rates). This disparity cannot be accounted for by variations in the degree of exposure to solvent in transition states (as measured by kinetic urea m values) and is, therefore, to be attributed mainly to differences in solvation-barrier contributions. This was supported by structure-energetics analyses of the transition states and by application of a novel procedure to estimate from experimental data the solvation-barrier impact at the entropy and free-energy levels. These analyses were actually performed with an extended protein set (including six small proteins plus seven variants of lipase from Thermomyces lanuginosus and spanning a wide range of activation parameters), allowing us to delineate the general trends of the solvation-barrier contributions. Overall, this work supports that proteins sharing the same structure and function but belonging to different organisms may show widely different solvation barriers, possibly as a result of different levels of the selection pressure associated with cooperativity, kinetic stability, and related factors.


Subject(s)
Trypanosoma/metabolism , Animals , Calorimetry, Differential Scanning , Kinetics , Models, Theoretical , Protein Denaturation , Solubility , Species Specificity , Thermodynamics
8.
Proteins ; 75(4): 977-89, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19089956

ABSTRACT

Microbial beta-1,4-galactanases are glycoside hydrolases belonging to family 53, which degrade galactan and arabinogalactan side chains in the hairy regions of pectin, a major plant cell wall component. They belong to the larger clan GH-A of glycoside hydrolases, which cover many different poly- and oligosaccharidase specificities. Crystallographic complexes of Bacillus licheniformis beta-1,4-galactanase and its inactive nucleophile mutant have been obtained with methyl-beta(1-->4)-galactotetraoside, providing, for the first time, information on substrate binding to the aglycone side of the beta-1,4-galactanase substrate binding groove. Using the experimentally determined subsites as a starting point, a beta(1-->4)-galactononaose was built into the structure and subjected to molecular dynamics simulations giving further insight into the residues involved in the binding of the polysaccharide from subsite -4 to +5. In particular, this analysis newly identified a conserved beta-turn, which contributes to subsites -2 to +3. This beta-turn is unique to family 53 beta-1,4-galactanases among all clan GH-A families that have been structurally characterized and thus might be a structural signature for endo-beta-1,4-galactanase specificity.


Subject(s)
Bacillus/enzymology , Galactans/chemistry , Glycoside Hydrolases/chemistry , Amino Acid Sequence , Binding Sites , Carbohydrate Conformation , Computer Simulation , Crystallography, X-Ray , Galactans/metabolism , Galactose/metabolism , Glycoside Hydrolases/metabolism , Models, Molecular , Molecular Sequence Data , Protein Binding , Sequence Alignment
9.
Proteins ; 70(1): 19-24, 2008 Jan 01.
Article in English | MEDLINE | ID: mdl-17640069

ABSTRACT

We have found that, contrary to naïve intuition, the degree of operational reversibility in the thermal denaturation of lipase from Thermomyces lanuginosa (an important industrial enzyme) in urea solutions is maximum when the protein is heated several degrees above the end of the temperature-induced denaturation transition. Upon cooling to room temperature, the protein seems to reach a state with enzymatic activity similar to that of the initial native state, but with higher denaturation temperature and radically different behavior in terms of susceptibility to irreversible denaturation. These results show that patterns of operational reversibility/irreversibility in protein denaturation may be more complex than the often-taken-for-granted, two-situation classification (reversible vs. irreversible). Furthermore, they are consistent with the possibility of existence of different native or native-like states separated by high kinetic barriers under native conditions and they suggest experimental procedures to reach and study such "alternative" native states.


Subject(s)
Proteins/chemistry , Calorimetry, Differential Scanning , Hot Temperature , Kinetics , Lipase/chemistry , Protein Denaturation
10.
J Biol Chem ; 282(26): 19177-89, 2007 Jun 29.
Article in English | MEDLINE | ID: mdl-17376777

ABSTRACT

The plant cell wall is a complex material in which the cellulose microfibrils are embedded within a mesh of other polysaccharides, some of which are loosely termed "hemicellulose." One such hemicellulose is xyloglucan, which displays a beta-1,4-linked d-glucose backbone substituted with xylose, galactose, and occasionally fucose moieties. Both xyloglucan and the enzymes responsible for its modification and degradation are finding increasing prominence, reflecting both the drive for enzymatic biomass conversion, their role in detergent applications, and the utility of modified xyloglucans for cellulose fiber modification. Here we present the enzymatic characterization and three-dimensional structures in ligand-free and xyloglucan-oligosaccharide complexed forms of two distinct xyloglucanases from glycoside hydrolase families GH5 and GH12. The enzymes, Paenibacillus pabuli XG5 and Bacillus licheniformis XG12, both display open active center grooves grafted upon their respective (beta/alpha)(8) and beta-jelly roll folds, in which the side chain decorations of xyloglucan may be accommodated. For the beta-jelly roll enzyme topology of GH12, binding of xylosyl and pendant galactosyl moieties is tolerated, but the enzyme is similarly competent in the degradation of unbranched glucans. In the case of the (beta/alpha)(8) GH5 enzyme, kinetically productive interactions are made with both xylose and galactose substituents, as reflected in both a high specific activity on xyloglucan and the kinetics of a series of aryl glycosides. The differential strategies for the accommodation of the side chains of xyloglucan presumably facilitate the action of these microbial hydrolases in milieus where diverse and differently substituted substrates may be encountered.


Subject(s)
Bacillus/enzymology , Glucans/metabolism , Glycoside Hydrolases/chemistry , Glycoside Hydrolases/metabolism , Xylans/metabolism , Bacillus/genetics , Cell Wall/metabolism , Cellulose/metabolism , Cloning, Molecular , Crystallization , Glucans/chemistry , Glycoside Hydrolases/genetics , Imaging, Three-Dimensional , Kinetics , Ligands , Structure-Activity Relationship , Substrate Specificity , Xylans/chemistry
11.
J Mol Biol ; 360(3): 715-24, 2006 Jul 14.
Article in English | MEDLINE | ID: mdl-16784752

ABSTRACT

The stability of several protein systems of interest has been shown to have a kinetic basis. Besides the obvious biotechnological implications, the general interest of understanding protein kinetic stability is emphasized by the fact that some emerging molecular approaches to the inhibition of amyloidogenesis focus on the increase of the kinetic stability of protein native states. Lipases are among the most important industrial enzymes. Here, we have studied the thermal denaturation of the wild-type form, four single-mutant variants and two highly stable, multiple-mutant variants of lipase from Thermomyces lanuginosa. In all cases, thermal denaturation was irreversible, kinetically controlled and conformed to the two-state irreversible model. This result supports that the novel molecular-dynamics-focused, directed-evolution approach involved in the preparation of the highly stable variants is successful likely because it addresses kinetic stability and, in particular, because heated molecular dynamics simulations possibly identify regions of disrupted native interactions in the transition state for irreversible denaturation. Furthermore, we find very large mutation effects on activation enthalpy and entropy, which were not accompanied by similarly large changes in kinetic urea m-value. From this we are led to conclude that these mutation effects are associated to some structural feature of the transition state for the irreversible denaturation process that is not linked to large changes in solvent accessibility. Recent computational studies have suggested the existence of solvation/desolvation barriers in at least some protein folding/unfolding processes. We thus propose that a solvation barrier (arising from the asynchrony between breaking of internal contacts and water penetration) may contribute to the kinetic stability of lipase from T. lanuginosa (and, possibly, to the kinetic stability of other proteins as well).


Subject(s)
Ascomycota/enzymology , Lipase/chemistry , Lipase/metabolism , Solvents/chemistry , Solvents/pharmacology , Kinetics , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Protein Denaturation/drug effects , Thermodynamics
12.
Biomol Eng ; 22(1-3): 21-30, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15857780

ABSTRACT

Modern enzyme development relies to an increasing extent on strategies based on diversity generation followed by screening for variants with optimised properties. In principle, these directed evolution strategies might be used for optimising any enzyme property, which can be screened for in an economically feasible way, even if the molecular basis of that property is not known. Stability is an interesting property of enzymes because (1) it is of great industrial importance, (2) it is relatively easy to screen for, and (3) the molecular basis of stability relates closely to contemporary issues in protein science such as the protein folding problem and protein folding diseases. Thus, engineering enzyme stability is of both commercial and scientific interest. Here, we review how directed evolution has contributed to the development of stable enzymes and to new insight into the principles of protein stability. Several recent examples are described. These examples show that directed evolution is an effective strategy to obtain stable enzymes, especially when used in combination with rational or semi-rational engineering strategies. With respect to the principles of protein stability, some important lessons to learn from recent efforts in directed evolution are (1) that there are many structural ways to stabilize a protein, which are not always easy to rationalize, (2) that proteins may very well be stabilized by optimizing their surfaces, and (3) that high thermal stability may be obtained without forfeiture of catalytic performance at low temperatures.


Subject(s)
Directed Molecular Evolution , Enzyme Stability/genetics , Enzymes/genetics , Animals , Catalysis , Directed Molecular Evolution/methods , Enzymes/chemistry , Humans , Protein Engineering/methods
13.
Acta Crystallogr D Biol Crystallogr ; 61(Pt 2): 190-3, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15681870

ABSTRACT

The enzymatic digestion of starch by alpha-amylases is one of the key biotechnological reactions of recent times. In the search for industrial biocatalysts, the family GH13 alpha-amylase BHA from Bacillus halmapalus has been cloned and expressed. The three-dimensional structure at 2.1 A resolution has been determined in complex with the (pseudo)tetrasaccharide inhibitor acarbose. Acarbose is found bound as a nonasaccharide transglycosylation product spanning the -6 to +3 subsites. Careful inspection of electron density suggests that the bound ligand could not have been formed through successive transglycosylations of acarbose and must also have featured maltose or maltooligosaccharides as an acceptor.


Subject(s)
Acarbose/chemistry , Bacillus/enzymology , Monosaccharides/chemistry , alpha-Amylases/chemistry , Carbohydrate Sequence , Models, Molecular , Molecular Sequence Data , Protein Conformation
14.
Acta Crystallogr D Biol Crystallogr ; 59(Pt 6): 1073-5, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12777778

ABSTRACT

Acetolactate decarboxylase has the unique ability to decarboxylate both enantiomers of acetolactate to give a single enantiomer of the decarboxylation product, (R)-acetoin. A gene coding for alpha-acetolactate decarboxylase from Bacillus brevis (ATCC 11031) was cloned and overexpressed in B. subtilis. The enzyme was purified in two steps to homogeneity prior to crystallization. Three different diffraction-quality crystal forms were obtained by the hanging-drop vapour-diffusion method using a number of screening conditions. The best crystal form is suitable for structural studies and was grown from solutions containing 20% PEG 2000 MME, 10 mM cadmium chloride and 0.1 M Tris-HCl pH 7.0. They grew to a maximum dimension of approximately 0.4 mm and belong to the trigonal space group P3(1,2)21, with unit-cell parameters a = 47.0, c = 198.9 A. A complete data set was collected to 2 A from a single native crystal using synchrotron radiation.


Subject(s)
Carboxy-Lyases/chemistry , Bacillus subtilis/enzymology , Carboxy-Lyases/biosynthesis , Carboxy-Lyases/isolation & purification , Cloning, Molecular , Crystallization , Crystallography, X-Ray
15.
Nat Biotechnol ; 20(12): 1251-5, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12426575

ABSTRACT

We describe synthetic shuffling, an evolutionary protein engineering technology in which every amino acid from a set of parents is allowed to recombine independently of every other amino acid. With the use of degenerate oligonucleotides, synthetic shuffling provides a direct route from database sequence information to functional libraries. Physical starting genes are unnecessary, and additional design criteria such as optimal codon usage or known beneficial mutations can also be incorporated. We performed synthetic shuffling of 15 subtilisin genes and obtained active and highly chimeric enzymes with desirable combinations of properties that we did not obtain by other directed-evolution methods.


Subject(s)
Amino Acids/genetics , Combinatorial Chemistry Techniques/methods , DNA Shuffling/methods , Protein Engineering/methods , Recombinant Proteins/genetics , Amino Acid Sequence , Amino Acids/chemistry , Bacillus subtilis/enzymology , Bacillus subtilis/genetics , Hydrogen-Ion Concentration , Molecular Sequence Data , Peptide Library , Sequence Alignment/methods , Sequence Analysis, Protein/methods
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