ABSTRACT
The exopolygalacturonase from Thermotoga maritima is the most thermoactive and thermostable pectinase known to date. Here we present its crystal structure at 2.05A resolution. High structural homology around the active site allowed us to propose a model for substrate binding, explaining the exo-cleavage activity and specificity for non-methylated saturated galacturonate at the non-reducing end. Furthermore, the structure reveals unique features that contribute to the formation of stable tetramers in solution. Such an oligomerization has not been observed before for polygalacturonases.
Subject(s)
Bacterial Proteins/chemistry , Glycoside Hydrolases/chemistry , Protein Multimerization , Thermotoga maritima/enzymology , Bacterial Proteins/metabolism , Catalytic Domain , Crystallization , Crystallography, X-Ray , Glycoside Hydrolases/metabolism , Hexuronic Acids/chemistry , Hexuronic Acids/metabolism , Hot Temperature , Models, Molecular , Protein Binding , Protein Structure, Quaternary , Protein Structure, Secondary , Protein Structure, Tertiary , Substrate SpecificityABSTRACT
Lipases are successfully applied in enantioselective biocatalysis. Most lipases contain a lid domain controlling access to the active site, but Bacillus subtilis Lipase A (LipA) is a notable exception: its active site is solvent exposed. To improve the enantioselectivity of LipA in the kinetic resolution of 1,2-O-isopropylidene-sn-glycerol (IPG) esters, we replaced a loop near the active-site entrance by longer loops originating from Fusarium solani cutinase and Penicillium purpurogenum acetylxylan esterase, thereby aiming to increase the interaction surface for the substrate. The resulting loop hybrids showed enantioselectivities inverted toward the desired enantiomer of IPG. The acetylxylan esterase-derived variant showed an inversion in enantiomeric excess (ee) from -12.9% to +6.0%, whereas the cutinase-derived variant was improved to an ee of +26.5%. The enantioselectivity of the cutinase-derived variant was further improved by directed evolution to an ee of +57.4%.
Subject(s)
Bacillus subtilis/enzymology , Lipase/chemistry , Lipase/metabolism , Protein Engineering/methods , Acetylesterase/chemistry , Acetylesterase/genetics , Amino Acid Sequence , Binding Sites , Carboxylic Ester Hydrolases/chemistry , Carboxylic Ester Hydrolases/genetics , Directed Molecular Evolution , Lipase/genetics , Models, Molecular , Molecular Sequence Data , Mutation , Protein Conformation , Stereoisomerism , Substrate SpecificityABSTRACT
Staphylococcus hyicus lipase differs from other bacterial lipases in its high phospholipase A1 activity. Here, we present the crystal structure of the S. hyicus lipase at 2.86 A resolution. The lipase is in an open conformation, with the active site partly covered by a neighbouring molecule. Ser124, Asp314 and His355 form the catalytic triad. The substrate-binding cavity contains two large hydrophobic acyl chain-binding pockets and a shallow and more polar third pocket that is capable of binding either a (short) fatty acid or a phospholipid head-group. A model of a phospholipid bound in the active site shows that Lys295 is at hydrogen bonding distance from the substrate's phosphate group. Residues Ser356, Glu292 and Thr294 hold the lysine in position by hydrogen bonding and electrostatic interactions. These observations explain the biochemical data showing the importance of Lys295 and Ser356 for phospholipid binding and phospholipase A1 activity.
Subject(s)
Phospholipases/chemistry , Phospholipases/metabolism , Staphylococcus/enzymology , Amino Acid Sequence , Conserved Sequence , Crystallography, X-Ray , Models, Molecular , Molecular Sequence Data , Phospholipases/genetics , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Alignment , Stereoisomerism , Substrate SpecificityABSTRACT
Phage display can be used as a protein-engineering tool for the selection of proteins with desirable binding properties from a library of mutants. Here we describe the application of this method for the directed evolution of Bacillus subtilis lipase A, an enzyme that has important properties for the preparation of the pharmaceutically relevant chiral compound 1,2-O-isopropylidene-sn-glycerol (IPG). PCR mutagenesis with spiked oligonucleotides was employed for saturation mutagenesis of a stretch of amino acids near the active site. After expression of these mutants on bacteriophages, dual selection with (S)-(+)- and (R)-(-)-IPG stereoisomers covalently coupled to enantiomeric phosphonate suicide inhibitors (SIRAN Sc and Rc inhibitors, respectively) was used for the isolation of variants with inverted enantioselectivity. The mutants were further characterised by determination of their Michaelis-Menten parameters. The 3D structures of the Sc and Rc inhibitor-lipase complexes were determined and provided structural insight into the mechanism of enantioselectivity of the enzyme. In conclusion, we have used phage display as a fast and reproducible method for the selection of Bacillus lipase A mutant enzymes with inverted enantioselectivity.
Subject(s)
Bacillus subtilis/enzymology , Bacterial Proteins/chemistry , Directed Molecular Evolution/methods , Enzyme Inhibitors/pharmacology , Peptide Library , Phosphates/pharmacology , Bacillus subtilis/drug effects , Bacterial Proteins/drug effects , Bacterial Proteins/genetics , Cloning, Molecular , Crystallization , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Hydrolysis , Models, Molecular , Molecular Structure , Mutation , Phosphates/chemistry , Protein Conformation , Protein Structure, Tertiary , Stereoisomerism , Structure-Activity RelationshipABSTRACT
Flavodoxin II from Azotobacter vinelandii is a "long-chain" flavodoxin and has one of the lowest E1 midpoint potentials found within the flavodoxin family. To better understand the relationship between structural features and redox potentials, the oxidized form of the C69A mutant of this flavodoxin was crystallized and its three-dimensional structure determined to a resolution of 2.25 A by molecular replacement. Its overall fold is similar to that of other flavodoxins, with a central five-stranded parallel beta-sheet flanked on either side by alpha-helices. An eight-residue insertion, compared with other long-chain flavodoxins, forms a short 3(10) helix preceding the start of the alpha3 helix. The flavin mononucleotide (FMN) cofactor is flanked by a leucine on its re face instead of the more conserved tryptophan, resulting in a more solvent-accessible FMN binding site and stabilization of the hydroquinone (hq) state. In particular the absence of a hydrogen bond to the N5 atom of the oxidized FMN was identified, which destabilizes the ox form, as well as an exceptionally large patch of acidic residues in the vicinity of the FMN N1 atom, which destabilizes the hq form. It is also argued that the presence of a Gly at position 58 in the sequence stabilizes the semiquinone (sq) form, as a result, raising the E2 value in particular.
Subject(s)
Azotobacter vinelandii/chemistry , Flavodoxin/chemistry , Flavodoxin/metabolism , Alanine/chemistry , Alanine/genetics , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Cysteine/chemistry , Cysteine/genetics , Flavin Mononucleotide/metabolism , Flavodoxin/genetics , Glycine/chemistry , Hydrogen Bonding , Leucine/chemistry , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Folding , Sequence Homology, Amino Acid , Structural Homology, Protein , Tryptophan/chemistryABSTRACT
The bipartite DNA-binding domain of Tc3 transposase, Tc3A, was crystallized in complex with its transposon recognition sequence. In the structure the two DNA-binding domains form structurally related helix-turn-helix (HTH) motifs. They both bind to the major groove on a single DNA oligomer, separated by a linker that interacts closely with the minor groove. The structure resembles that of the transcription factor Pax6 DNA-binding domain, but the relative orientation of the HTH-domain is different. The DNA conformation is distorted, characterized by local narrowing of the minor groove and bends at both ends. The protein-DNA recognition takes place through base and backbone contacts, as well as shape-recognition of the distortions in the DNA. Charged interactions are primarily found in the N-terminal domain and the linker indicating that these may form the initial contact area. Two independent dimer interfaces could be relevant for bringing together transposon ends and for binding to a direct repeat site in the transposon end. In contrast to the Tn5 synaptic complex, the two Tc3A DNA-binding domains bind to a single Tc3 transposon end.
Subject(s)
DNA Transposable Elements , DNA-Binding Proteins/chemistry , Transposases/chemistry , Base Sequence , Binding Sites , DNA/chemistry , DNA/metabolism , DNA-Binding Proteins/metabolism , Dimerization , Models, Molecular , Molecular Sequence Data , Protein Structure, Tertiary , Transposases/metabolismABSTRACT
Endopolygalacturonase I is a processive enzyme, while the 60% sequence identical endopolygalacturonase II is not. The 1.70 A resolution crystal structure of endopolygalacturonase I reveals a narrowed substrate binding cleft. In addition, Arg96, a residue in this cleft previously shown to be critical for processivity, interacts with the substrate mimics glycerol and sulfate in several well-defined conformations in the six molecules in the asymmetric unit. From this we conclude that both Arg96 and the narrowed substrate binding cleft contribute to retaining the substrate while it moves through the active site after a cleavage event has occurred.
Subject(s)
Aspergillus niger/enzymology , Polygalacturonase/chemistry , Arginine/chemistry , Binding Sites , Carbohydrate Sequence , Crystallography, X-Ray , Glycerol/metabolism , Glycoside Hydrolases/chemistry , Glycoside Hydrolases/genetics , Glycosylation , Models, Molecular , Molecular Sequence Data , Polygalacturonase/genetics , Protein Structure, Secondary , Sulfates/metabolismABSTRACT
Cytochrome c(551) from Pseudomonas aeruginosa is a monomeric redox protein of 82 amino-acid residues, involved in dissimilative denitrification as the physiological electron donor of cd(1) nitrite reductase. The distribution of charged residues on the surface of c(551) is very anisotropic: one side is richer in acidic residues whereas the other shows a ring of positive side chains, mainly lysines, located at the border of an hydrophobic patch which surrounds the heme crevice. In order to map in cytochrome c(551) the surface involved in electron transfer, we have introduced specific mutations in three residues belonging to the hydrophobic patch, namely Val23-->Asp, Pro58-->Ala and Ile59-->Glu. The effect of these mutations was analyzed studying both the self-exchange rate and the electron-transfer activity towards P. aeruginosa cd(1) nitrite reductase, the physiological partner and P. aeruginosa azurin, a copper protein often used as a model redox partner in vitro. Our results show that introduction of a negative charge in the hydrophobic patch severely hampers both homonuclear and heteronuclear electron transfer.
