Gertie van Pouderoyen

Structural insights into the processivity of endopolygalacturonase I from Aspergillus niger

Endopolygalacturonase I is a processive enzyme, while the 60% sequence identical endopolygalacturonase II is not. The 1.70 Å 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.

Directed Evolution of Bacillus subtilis Lipase A by Use of Enantiomeric Phosphonate Inhibitors: Crystal Structures and Phage Display Selection

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.

A crystallographic study of Cys69Ala flavodoxin II from Azotobacter vinelandii: Structural determinants of redox potential

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 Å by molecular replacement. Its overall fold is similar to that of other flavodoxins, with a central five‐stranded parallel β‐sheet flanked on either side by α‐helices. An eight‐residue insertion, compared with other long‐chain flavodoxins, forms a short 310 helix preceding the start of the α3 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.

Lipolytic enzymes LipA and LipB from Bacillus subtilis differ in regulation of gene expression, biochemical properties, and three-dimensional structure.

Bacillus subtilis secretes the lipolytic enzymes LipA and LipB. We show here that they are differentially expressed depending on the composition of the growth medium: LipA is produced in rich and in minimal medium, whereas LipB is present only in rich medium. A comparison of biochemical characteristics revealed that LipB is thermostable at pH 11 but becomes thermolabile at pH 5. However, construction of a variant carrying the substitution A76G in the conserved lipase pentapeptide reversed these effects. The atomic coordinates from the LipA crystal structure were used to build a three‐dimensional structural model of LipB, which revealed that 43 out of 45 residues different from LipA are surface‐located allowing to rationalize the differences observed in the substrate preferences of the two enzymes.

Biochemical properties and three-dimensional structures of two extracellular lipolytic enzymes from Bacillus subtilis

This article reviews our present knowledge on the extracellular lipolytic enzymes LipA and LipB from Bacillus subtilis. Growth of B. subtilis to the late logarithmic growth phase results in a total lipolytic activity of 12–18 units per liter of culture supernatant. Immunodetection with LipA- and LipB-specific antibodies indicated a differential expression of both lipolytic enzymes depending on the composition of the growth medium. LipA was produced in rich and in minimal medium, whereas LipB was present only in rich medium. The lipA and lipB genes were cloned and overexpressed in B. subtilis and Escherichia coli, the corresponding proteins purified to electrophoretic homogeneity and their substrate specificities, pH- and temperature stabilities were determined. The active site residue Ser78 of LipB is located in the consensus sequence Ala–X–Ser–X–Gly where the alanine replaces a glycine found in most of the bacterial lipases. The role of this Ala-residue was investigated by constructing LipB variant A76G thereby restoring the canonical lipase consensus motif. When compared with wild-type LipB this variant showed a markedly reduced thermostability at pH 11 but an increased stability at pH 5–7. These findings were rationalized by building a three-dimensional structural model of LipB using the atomic coordinates of the LipA crystal structure, which was solved recently. The LipB model structure revealed that 43 out of 45 residues, which are different from LipA, were located on the surface of LipB. The surface-exposed amino acids including those located at the rim of the active site cleft may cause the differences in specific activities between LipA and LipB.

Structure-function correlation of intramolecular electron transfer in wild type and single-site mutated azurins

Abstract Intramolecular electron transfer (ET) between the Cys3-Cys26 radical ion (RSSR − ) produced pulse radiolytically and the Cu(II) ion has been studied in four wild type and nine different single site mutants of the blue single-copper protein, azurin. This enabled examination of the rate of this intramolecular ET as a function of driving force and the nature of the medium separating the electron donor and acceptor. Using a tunneling pathway model for ET from donor (RSSR − ) to acceptor (Cu[II]) through a combination of covalent bonds, hydrogen bonds, and space (van der Waals contact) jumps, the electronic coupling decays for protein mediated ET were calculated and potential pathways operating within the different azurins could be predicted. The rates of intramolecular ET and activation parameters for the above azurins correlate well with pathway distance and driving force as predicted by the Marcus theory, using a through-bond ET mechanism.

Pseudomonas aeruginosa cytochrome C551: Probing the role of the hydrophobic patch in electron transfer

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.

Loop grafting of Bacillus subtilis lipase A : Inversion of enantioselectivity

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%.

The crystal structure of a hyperthermoactive exopolygalacturonase from Thermotoga maritima reveals a unique tetramer

The exopolygalacturonase from Thermotoga maritima is the most thermoactive and thermostable pectinase known to date. Here we present its crystal structure at 2.05 Å 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.

Cysteine ligand vibrations are responsible for the complex resonance Raman spectrum of azurin

Abstract In the redox center of azurin, the Cu(II) is strongly coordinated to one thiolate S from Cys 112 and two imidazole Ns from His 46 and 117. This site yields a complex resonance Raman (RR) spectrum with >20 vibrational modes between 200 and 1500 cm–1. We have investigated the effects of ligand-selective isotope replacements on the RR spectrum of Pseudomonas aeruginosa azurin to determine the relative spectral contribution from each of the copper ligands. Growth on 34S-sulfate labels the cysteine ligand and allows the identification of a cluster of bands with Cu–S(Cys) stretching character between 370 and 430 cm–1 whose frequencies are consistent with the trigonal or distorted tetrahedral coordination in type 1 sites. In type 2 copper-cysteinate sites, the lower ν (Cu–S) frequencies between 260 and 320 cm–1 are consistent with square-planar coordination. Addition of exogenous15N-labeled imidazole or histidine to the His117Gly mutant generates type 1 or type 2 sites, respectively. Because neither the above nor the His46Gly mutant reconstituted with 15N-imidazole exhibits significant isotope dependence, the histidine ligands can be ruled out as important contributors to the RR spectrum. Instead, a variety of evidence, including extensive isotope shifts upon global substitution with 15N, suggests that the multiple RR modes of azurin are due principally to vibrations of the cysteine ligand. These are resonance-enhanced through kinematic coupling with the Cu–S stretch in the ground state or through an excited-state A-term mechanism involving a Cu-cysteinate chromophore that extends into the peptide backbone.