A 118 kDa fragment, comprising the catalytic domain and four other domains, of the glucansucrase GTFA from L. reuteri 121, which synthesizes α-glucans with both α-1,6- and α-1,4-glycosidic linkages, was crystallized. The weakly diffracting crystals, which contained 85% solvent, were used to determine the structure at 3.6 Å resolution.
The reuteransucrase GTFA from Lactobacillus reuteri 121, which belongs to glycosyl hydrolase family GH70, synthesizes branched α-glucans with both α-1,6- and α-1,4-glycosidic linkages (reuteran) from sucrose. The crystal structure of GTFA-ΔN, a 118 kDa fragment of GTFA comprising residues 745–1763 and including the catalytic domain, was determined at 3.6 Å resolution by molecular replacement. The crystals have large solvent channels and an unusually high solvent content of 85%. GTFA-ΔN has the same domain arrangement and domain topologies as observed in previously determined GH70 glucansucrase structures. The architecture of the GTFA-ΔN active site and binding pocket confirms that glucansucrases have a conserved substrate specificity for sucrose. However, this first crystal structure of an α-1,6/α-1,4-specific glucansucrase shows that residues from conserved sequence motif IV (1128–1136 in GTFA-ΔN) contribute to the acceptor-binding subsites and that they display differences compared with other structurally characterized glucansucrases. In particular, the structure clarifies the importance of residues following the transition-state stabilizer for product specificity, and especially residue Asn1134, which is in a position to interact with sugar units in acceptor subsite +2.
lactic acid bacteria; glucansucrase; reuteransucrase
3-Ketosteroid Δ1-dehydrogenase from Rhodococcus erythropolis SQ1 was successfully crystallized and its initial structure was solved.
3-Ketosteroid Δ1-dehydrogenase plays a crucial role in the early steps of steroid degradation by introducing a double bond between the C1 and C2 atoms of the A-ring of its 3-ketosteroid substrates. The 3-ketosteroid Δ1-dehydrogenase from Rhodococcus erythropolis SQ1, a 56 kDa flavoprotein, was crystallized using the sitting-drop vapour-diffusion method at room temperature. The crystals grew in various buffers over a wide pH range (from pH 5.5 to 10.5), but the best crystallization condition consisted of 2%(v/v) PEG 400, 0.1 M HEPES pH 7.5, 2.0 M ammonium sulfate. A native crystal diffracted X-rays to 2.0 Å resolution. It belonged to the primitive orthorhombic space group P212121, with unit-cell parameters a = 107.4, b = 131.6, c = 363.2 Å, and contained eight molecules in the asymmetric unit. The initial structure of the enzyme was solved using multi-wavelength anomalous dispersion (MAD) data collected from a Pt-derivatized crystal.
3-ketosteroid Δ1-dehydrogenase; Rhodococcus erythropolis SQ1
The gene for 3-ketosteroid Δ4-(5α)-dehydrogenase from R. jostii RHA1 was cloned and overexpressed in E. coli and the protein product was purified and crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to space group C2221 and diffraction data were collected to a resolution of 1.6 Å.
3-Ketosteroid dehydrogenases are flavoproteins which play key roles in steroid ring degradation. The enzymes are abundantly present in actinobacteria, including the catabolic powerhouse Rhodococcus jostii and the pathogenic species R. equi and Mycobacterium tuberculosis. The gene for 3-ketosteroid Δ4-(5α)-dehydrogenase [Δ4-(5α)-KSTD] from R. jostii RHA1 was cloned and overexpressed in Escherichia coli. His-tagged Δ4-(5α)-KSTD enzyme was purified by Ni2+–NTA affinity chromatography, anion-exchange chromatography and size-exclusion chromatography and was crystallized using the hanging-drop vapour-diffusion method. Seeding greatly improved the number of crystals obtained. The crystals belonged to space group C2221, with unit-cell parameters a = 99.2, b = 114.3, c = 110.2 Å. Data were collected to a resolution of 1.6 Å.
3-ketosteroid dehydrogenases; Rhodococcus jostii RHA1
Lactococcus lactis expresses the homologous glucosaminidases AcmB, AcmC, AcmA and AcmD. The latter two have three C-terminal LysM repeats for peptidoglycan binding. AcmD has much shorter intervening sequences separating the LysM repeats and a lower iso-electric point (4.3) than AcmA (10.3). Under standard laboratory conditions AcmD was mainly secreted into the culture supernatant. An L. lactis acmAacmD double mutant formed longer chains than the acmA single mutant, indicating that AcmD contributes to cell separation. This phenotype could be complemented by plasmid-encoded expression of AcmD in the double mutant. No clear difference in cellular lysis and protein secretion was observed between both mutants. Nevertheless, overexpression of AcmD resulted in increased autolysis when AcmA was present (as in the wild type strain) or when AcmA was added to the culture medium of an AcmA-minus strain. Possibly, AcmD is mainly active within the cell wall, at places where proper conditions are present for its binding and catalytic activity. Various fusion proteins carrying either the three LysM repeats of AcmA or AcmD were used to study and compare their cell wall binding characteristics. Whereas binding of the LysM domain of AcmA took place at pHs ranging from 4 to 8, LysM domain of AcmD seems to bind strongest at pH 4.
By selective enrichment, we isolated a bacterium that can use β-phenylalanine as a sole nitrogen source. It was identified by 16S rRNA gene sequencing as a strain of Variovorax paradoxus. Enzyme assays revealed an aminotransferase activity. Partial genome sequencing and screening of a cosmid DNA library resulted in the identification of a 1,302-bp aminotransferase gene, which encodes a 46,416-Da protein. The gene was cloned and overexpressed in Escherichia coli. The recombinant enzyme was purified and showed a specific activity of 17.5 U mg−1 for (S)-β-phenylalanine at 30°C and 33 U mg−1 at the optimum temperature of 55°C. The β-specific aminotransferase exhibits a broad substrate range, accepting ortho-, meta-, and para-substituted β-phenylalanine derivatives as amino donors and 2-oxoglutarate and pyruvate as amino acceptors. The enzyme is highly enantioselective toward (S)-β-phenylalanine (enantioselectivity [E], >100) and derivatives thereof with different substituents on the phenyl ring, allowing the kinetic resolution of various racemic β-amino acids to yield (R)-β-amino acids with >95% enantiomeric excess (ee). The crystal structures of the holoenzyme and of the enzyme in complex with the inhibitor 2-aminooxyacetate revealed structural similarity to the β-phenylalanine aminotransferase from Mesorhizobium sp. strain LUK. The crystal structure was used to rationalize the stereo- and regioselectivity of V. paradoxus aminotransferase and to define a sequence motif with which new aromatic β-amino acid-converting aminotransferases may be identified.
Mushroom tyrosinase was crystallized in two different space groups. The crystals diffracted to 3.0 Å resolution (P21) or 2.6 Å resolution (P21212).
Tyrosinase catalyzes the conversion of tyrosine to dihydroxyphenylalanine quinone, which is the main precursor for the biosynthesis of melanin. The enzyme from Agaricus bisporus, the common button mushroom, was purified and crystallized in two different space groups. Crystals belonging to space group P21 (unit-cell parameters a = 104.2, b = 105.0, c = 119.1 Å, β = 110.6°, four molecules per asymmetric unit) diffracted to 3.0 Å resolution. Crystals belonging to space group P21212 (unit-cell parameters a = 104.0, b = 104.5, c = 108.4 Å, two molecules per asymmetric unit) diffracted to 2.6 Å resolution. It was essential to include 5 mM HoCl3 in all crystallization conditions in order to obtain well diffracting crystals.
tyrosinase; Agaricus bisporus
The major murein and pseudomurein cell wall-binding domains, i.e., the Lysin Motif (LysM) (Pfam PF01476) and pseudomurein cell wall-binding (PMB) (Pfam PF09373) motif, respectively, were genetically fused. The fusion protein is capable of binding to both murein- and pseudomurein-containing cell walls. In addition, it also binds to chitin, the major polymer of fungal cell walls. Binding is influenced by pH and occurs at a pH close to the pI of the binding protein. Functional studies on truncated versions of the fusion protein revealed that murein and chitin binding is provided by the LysM domain, while binding to pseudomurein is achieved through the PMB domain.
Murein; Pseudomurein; Chitin; Domain
Aptamers are oligonucleotide ligands, either RNA or ssDNA, selected for high-affinity binding to molecular targets, such as small organic molecules, proteins or whole microorganisms. While reports of new aptamers are numerous, characterization of their specific interaction is often restricted to the affinity of binding (KD). Over the years, crystal structures of aptamer-protein complexes have only scarcely become available. Here we describe some relevant technical issues about the process of crystallizing aptamer-protein complexes and highlight some biochemical details on the molecular basis of selected aptamer-protein interactions. In addition, alternative experimental and computational approaches are discussed to study aptamer-protein interactions.
X-ray crystallography; aptamer; interaction; RNA/DNA-protein complex
The cell wall, a major barrier protecting cells from their environment, is an essential compartment of both bacteria and archaea. It protects the organism from internal turgor pressure and gives a defined shape to the cell. The cell wall serves also as an anchoring surface for various proteins and acts as an adhesion platform for bacteriophages. The walls of bacteria and archaea are mostly composed of murein and pseudomurein, respectively. Cell wall binding domains play a crucial role in the non-covalent attachment of proteins to cell walls. Here, we give an overview of the similarities and differences in the biochemical and functional properties of the two major murein and pseudomurein cell wall binding domains, i.e., the Lysin Motif (LysM) domain (Pfam PF01476) and the pseudomurein binding (PMB) domain (Pfam PF09373) of bacteria and archaea, respectively.
Murein; Pseudomurein; Motifs and domains
We have biochemically and functionally characterized the pseudomurein cell wall-binding (PMB) domain that is present at the C-terminus of the Surface (S)-layer protein MTH719 from Methanothermobacter thermautotrophicus. Chemical denaturation of the protein with guanidinium hydrochloride occurred at 3.8 M. A PMB-GFP fusion protein not only binds to intact pseudomurein of methanogenic archaea, but also to spheroplasts of lysozyme-treated bacterial cells. This binding is pH dependent. At least two of the three motifs that are present in the domain are necessary for binding. Limited proteolysis revealed a possible cleavage site in the spacing sequence between motifs 1 and 2 of the PMB domain, indicating that the motif region itself is protected from proteases.
The subtilisin-like serine protease carnein was isolated from the latex of the plant I. carnea, purified and crystallized by the hanging-drop vapour-diffusion method. A diffraction data set was collected to 2.0 Å resolution in-house from a single crystal at 110 K.
Carnein is an 80 kDa subtilisin-like serine protease from the latex of the plant Ipomoea carnea which displays an exceptional resistance to chemical and thermal denaturation. In order to obtain the first crystal structure of a plant subtilisin and to gain insight into the structural determinants underlying its remarkable stability, carnein was isolated from I. carnea latex, purified and crystallized by the hanging-drop vapour-diffusion method. A data set was collected to 2.0 Å resolution in-house from a single crystal at 110 K. The crystals belonged to the trigonal space group P3121 or P3221, with unit-cell parameters a = b = 126.9, c = 84.6 Å, α = β = 90, γ = 120°. Assuming the presence of one molecule per asymmetric unit, the Matthews coefficient is 2.46 Å3 Da−1, corresponding to a solvent content of 50%. Structure determination of the enzyme is in progress.
carnein; serine proteases; subtilisin
PeiW (UniProtKB Q7LYX0) and PeiP (UniProtKB Q77WJ4) are the two major pseudomurein endoisopeptidases (Pei) that are known to cleave pseudomurein cell-wall sacculi of the members of the methanogenic orders Methanobacteriales and Methanopyrales. Both enzymes, originating from prophages specific for some methanogenic archaeal species, hydrolyze the ϵ(Ala)-Lys bond of the peptide linker between adjacent pseudomurein layers. Because lysozyme is not able to cleave the pseudomurein cell wall, the enzymes are used in protoplast preparation and in DNA isolation from pseudomurein cell-wall-containing methanogens. Moreover, PeiW increases the probe permeability ratio and enables fluorescence in situ hybridization (FISH) and catalyzed reporter deposition (CARD-) FISH experiments to be performed on these methanogens.
The β-xylosidase was crystallized using PEG 6000 as precipitant. 5% PEG 6000 yielded bipyramid-shaped tetragonal crystals diffracting to 1.55 Å resolution, and 13% PEG 6000 gave rectangular monoclinic crystals diffracting to 1.80 Å resolution.
The main enzymes involved in xylan-backbone hydrolysis are endo-1,4-β-xylanase and β-xylosidase. β-Xylosidase converts the xylo-oligosaccharides produced by endo-1,4-β-xylanase into xylose monomers. The β-xylosidase from the thermophilic Geobacillus thermoleovorans IT-08, a member of glycoside hydrolase family 43, was crystallized at room temperature using the hanging-drop vapour-diffusion method. Two crystal forms were observed. Bipyramid-shaped crystals belonging to space group P43212, with unit-cell parameters a = b = 62.53, c = 277.4 Å diffracted to 1.55 Å resolution. The rectangular crystals belonged to space group P21, with unit-cell parameters a = 57.94, b = 142.1, c = 153.9 Å, β = 90.5°, and diffracted to 1.80 Å resolution.
β-xylosidase; glycoside hydrolase family 43; Geobacillus thermoleovorans IT-08
A thermostable esterase (EstA) from Thermotoga maritima was cloned and purified. Crystals of EstA and its selenomethionine derivative were grown and diffract to beyond 2.6 Å resolution at 100 K using synchrotron radiation.
A predicted esterase (EstA) with an unusual new domain from the hyperthermophilic bacterium Thermotoga maritima has been cloned and overexpressed in Escherichia coli. The purified protein was crystallized by the hanging-drop vapour-diffusion technique in the presence of lithium sulfate and polyethylene glycol 8000. Selenomethionine-substituted EstA crystals were obtained under the same conditions and three different-wavelength data sets were collected to 2.6 Å resolution. The crystal belongs to space group H32, with unit-cell parameters a = b = 130.2, c = 306.2 Å. There are two molecules in the asymmetric unit, with a V
M of 2.9 Å3 Da−1 and 58% solvent content.
esterases; Thermotoga maritima
E. coli bacterioferritin was crystallized in a novel crystal form from different conditions and the structure was solved. The crystals belonged to space group P213 and diffracted to a resolution of 2.5 Å.
Escherichia coli bacterioferritin was serendipitously crystallized in a novel cubic crystal form and its structure could be determined to 2.5 Å resolution despite a high degree of merohedral twinning. This is the first report of crystallographic data on ‘as-isolated’ E. coli bacterioferritin. The ferroxidase active site contains positive difference density consistent with two metal ions that had co-purified with the protein. X-ray fluorescence studies suggest that the metal composition is different from that of previous structures and is a mix of zinc and native iron ions. The ferroxidase-centre configuration displays a similar flexibility as previously noted for other bacterioferritins.
Escherichia coli bacterioferritin; iron storage and homeostasis; ferroxidase; merohedral twinning
Haloalcohol dehalogenases are bacterial enzymes that cleave the carbon-halogen bond in short aliphatic vicinal haloalcohols, like 1-chloro-2,3-propanediol, some of which are recalcitrant environmental pollutants. They use a conserved Ser-Tyr-Arg catalytic triad to deprotonate the haloalcohol oxygen, which attacks the halogen-bearing carbon atom, producing an epoxide and a halide ion. Here, we present the X-ray structure of the haloalcohol dehalogenase HheAAD2 from Arthrobacter sp. strain AD2 at 2.0-Å resolution. Comparison with the previously reported structure of the 34% identical enantioselective haloalcohol dehalogenase HheC from Agrobacterium radiobacter AD1 shows that HheAAD2 has a similar quaternary and tertiary structure but a much more open substrate-binding pocket. Docking experiments reveal that HheAAD2 can bind both enantiomers of the haloalcohol substrate 1-p-nitrophenyl-2-chloroethanol in a productive way, which explains the low enantiopreference of HheAAD2. Other differences are found in the halide-binding site, where the side chain amino group of Asn182 is in a position to stabilize the halogen atom or halide ion in HheAAD2, in contrast to HheC, where a water molecule has taken over this role. These results broaden the insight into the structural determinants that govern reactivity and selectivity in the haloalcohol dehalogenase family.