Differences in crystallization of two LinB variants from Sphingobium japonicum UT26

Haloalkane dehalogenases are microbial enzymes that convert a broad range of halogenated aliphatic compounds to their corresponding alcohols by the hydrolytic mechanism. These enzymes play an important role in the biodegradation of various environmental pollutants. Haloalkane dehalogenase LinB isolated from a soil bacterium Sphingobium japonicum UT26 has a relatively broad substrate specificity and can be applied in bioremediation and biosensing of environmental pollutants. The LinB variants presented here, LinB32 and LinB70, were constructed with the goal of studying the effect of mutations on enzyme functionality. In the case of LinB32 (L117W), the introduced mutation leads to blocking of the main tunnel connecting the deeply buried active site with the surrounding solvent. The other variant, LinB70 (L44I, H107Q), has the second halide-binding site in a position analogous to that in the related haloalkane dehalogenase DbeA from Bradyrhizobium elkanii USDA94. Both LinB variants were successfully crystallized and full data sets were collected for native enzymes as well as their complexes with the substrates 1,2-dibromoethane (LinB32) and 1-bromobutane (LinB70) to resolutions ranging from 1.6 to 2.8 Å. The two mutants crystallize differently from each other, which suggests that the mutations, although deep inside the molecule, can still affect the protein crystallizability

Crystallization and preliminary crystallographic characterization of the iron regulated outer membrane lipoprotein FrpD from Neisseria meningitidis

Fe-regulated protein D (FrpD) is a Neisseria meningitidis outer membrane lipoprotein that may be involved in the anchoring of the secreted repeat in toxins (RTX) protein FrpC to the outer bacterial membrane. However, the function and biological roles of the FrpD and FrpC proteins remain unknown. Native and selenomethionine-substituted variants of recombinant FrpD(43–271) protein were crystallized using the sitting-drop vapour-diffusion method. Diffraction data were collected to a resolution of 2.25 Å for native FrpD(43–271) protein and to a resolution of 2.00 Å for selenomethionine-substituted FrpD(43–­271) (SeMet FrpD(43–271)) protein. The crystals of native FrpD(43–271) protein belonged to the hexagonal space group P6(2) or P6(4), while the crystals of SeMet FrpD(43–271) protein belonged to the primitive orthorhombic space group P2(1)2(1)2(1)

Cloning, expression, purification, crystallization and preliminary X ray diffraction analysis of AHP2, a signal transmitter protein from Arabidopsis thaliana

Histidine-containing phosphotransfer proteins from Arabidopsis thaliana (AHP1–5) act as intermediates between sensor histidine kinases and response regulators in a signalling system called multi-step phosphorelay (MSP). AHP proteins mediate and potentially integrate various MSP-based signalling pathways (e.g. cytokinin or osmosensing). However, structural information about AHP proteins and their importance in MSP signalling is still lacking. To obtain a deeper insight into the structural basis of AHP-mediated signal transduction, the three-dimensional structure of AHP2 was determined. The AHP2 coding sequence was cloned into pRSET B expression vector, enabling production of AHP2 fused to an N-terminal His tag. AHP2 was expressed in soluble form in Escherichia coli strain BL21 (DE3) pLysS and then purified to homogeneity using metal chelate affinity chromatography and anion-exchange chromatography under reducing conditions. Successful crystallization in a buffer which was optimized for thermal stability yielded crystals that diffracted to 2.5 Å resolution

Crystallographic analysis of new psychrophilic haloalkane dehalogenases DpcA from Psychrobacter cryohalolentis K5 and DmxA from Marinobacter sp ELB17

Haloalkane dehalogenases are hydrolytic enzymes with a broad range of potential practical applications such as biodegradation, biosensing, biocatalysis and cellular imaging. Two newly isolated psychrophilic haloalkane dehalogenases exhibiting interesting catalytic properties, DpcA from Psychrobacter cryohalolentis K5 and DmxA from Marinobacter sp. ELB17, were purified and used for crystallization experiments. After the optimization of crystallization conditions, crystals of diffraction quality were obtained. Diffraction data sets were collected for native enzymes and complexes with selected ligands such as 1-bromohexane and 1,2-dichloroethane to resolutions ranging from 1.05 to 2.49 Å

Structural basis of the interaction between the putative adhesion involved and iron regulated FrpD and FrpC proteins of Neisseria meningitidis

The iron-regulated protein FrpD from $Neisseria$ $meningitidis$ is an outer membrane lipoprotein that interacts with very high affinity ($K_d \sim 0.2$ nM) with the N-terminal domain of FrpC, a Type I-secreted protein from the $\underline{R}$epeat in $\underline{T}$o$\underline{X}$in (RTX) protein family. In the presence of Ca$^{2+}$, FrpC undergoes Ca$^{2+}$-dependent protein trans-splicing that includes an autocatalytic cleavage of the Asp$_{414}$-Pro$_{415}$ peptide bond and formation of an Asp$_{414}$-Lys isopeptide bond. Here, we report the high-resolution structure of FrpD and describe the structure-function relationships underlying the interaction between FrpD and FrpC$_{1-414}$. We identified FrpD residues involved in FrpC$_{1-414}$ binding, which enabled localization of FrpD within the low-resolution SAXS model of the FrpD-FrpC$_{1-414}$ complex. Moreover, the trans-splicing activity of FrpC resulted in covalent linkage of the FrpC$_{1-414}$ fragment to plasma membrane proteins of epithelial cells in vitro, suggesting that formation of the FrpD-FrpC$_{1-414}$ complex may be involved in the interaction of meningococci with the host cell surface

Engineering a de Novo Transport Tunnel

Transport of ligands between buried active sites and bulk solvent is a key step in the catalytic cycle of many enzymes. The absence of evolutionary optimized transport tunnels is an important barrier limiting the efficiency of biocatalysts prepared by computational design. Creating a structurally defined and functional “hole” into the protein represents an engineering challenge. Here we describe the computational design and directed evolution of a de novo transport tunnel in haloalkane dehalogenase. Mutants with a blocked native tunnel and newly opened auxiliary tunnel in a distinct part of the structure showed dramatically modified properties. The mutants with blocked tunnels acquired specificity never observed with native family members: up to 32 times increased substrate inhibition and 17 times reduced catalytic rates. Opening of the auxiliary tunnel resulted in specificity and substrate inhibition similar to those of the native enzyme and the most proficient haloalkane dehalogenase reported to date (<i>k</i>_cat = 57 s^–1 with 1,2-dibromoethane at 37 °C and pH 8.6). Crystallographic analysis and molecular dynamics simulations confirmed the successful introduction of a structurally defined and functional transport tunnel. Our study demonstrates that, whereas we can open the transport tunnels with reasonable proficiency, we cannot accurately predict the effects of such change on the catalytic properties. We propose that one way to increase efficiency of an enzyme is the direct its substrates and products into spatially distinct tunnels. The results clearly show the benefits of enzymes with de novo transport tunnels, and we anticipate that this engineering strategy will facilitate the creation of a wide range of useful biocatalysts

Structural and Functional Analysis of Novel Haloalkane Dehalogenase with Two Halide Binding Sites

The crystal structure of the novel haloalkane dehalogenase DbeA fromBradyrhizobium elkaniiUSDA94 revealed the presence of two chloride ions buried in the protein interior. The first halide-binding site is involved in substrate binding and is present in all structurally characterized haloalkane dehalogenases. The second halide-binding site is unique to DbeA. To elucidate the role of the second halide-binding site in enzyme functionality, a two-point mutant lacking this site was constructed and characterized. These substitutions resulted in a shift in the substrate-specificity class and were accompanied by a decrease in enzyme activity, stability and the elimination of substrate inhibition. The changes in enzyme catalytic activity were attributed to deceleration of the rate-limiting hydrolytic step mediated by the lower basicity of the catalytic histidine.</jats:p