Dong-Ju You

Crystal structure of a family I.3 lipase from Pseudomonas sp. MIS38 in a closed conformation

The crystal structure of a family I.3 lipase from Pseudomonas sp. MIS38 in a closed conformation was determined at 1.5 Å resolution. This structure highly resembles that of Serratia marcescens LipA in an open conformation, except for the structures of two lids. Lid1 is anchored by a Ca2+ ion (Ca1) in an open conformation, but lacks this Ca1 site and greatly changes its structure and position in a closed conformation. Lid2 forms a helical hairpin in an open conformation, but does not form it and covers the active site in a closed conformation. Based on these results, we discuss on the lid‐opening mechanism.

Crystal structure and thermodynamic and kinetic stability of metagenome-derived LC-cutinase.

The crystal structure of metagenome-derived LC-cutinase with polyethylene terephthalate (PET)-degrading activity was determined at 1.5 Å resolution. The structure strongly resembles that of Thermobifida alba cutinase. Ser165, Asp210, and His242 form the catalytic triad. Thermal denaturation and guanidine hydrochloride (GdnHCl)-induced unfolding of LC-cutinase were analyzed at pH 8.0 by circular dichroism spectroscopy. The midpoint of the transition of the thermal denaturation curve, T1/2, and that of the GdnHCl-induced unfolding curve, Cm, at 30 °C were 86.2 °C and 4.02 M, respectively. The free energy change of unfolding in the absence of GdnHCl, ΔG(H2O), was 41.8 kJ mol(-1) at 30 °C. LC-cutinase unfolded very slowly in GdnHCl with an unfolding rate, ku(H2O), of 3.28 × 10(-6) s(-1) at 50 °C. These results indicate that LC-cutinase is a kinetically robust protein. Nevertheless, the optimal temperature for the activity of LC-cutinase toward p-nitrophenyl butyrate (50 °C) was considerably lower than the T1/2 value. It increased by 10 °C in the presence of 1% polyethylene glycol (PEG) 1000. It also increased by at least 20 °C when PET was used as a substrate. These results suggest that the active site is protected from a heat-induced local conformational change by binding of PEG or PET. LC-cutinase contains one disulfide bond between Cys275 and Cys292. To examine whether this disulfide bond contributes to the thermodynamic and kinetic stability of LC-cutinase, C275/292A-cutinase without this disulfide bond was constructed. Thermal denaturation studies and equilibrium and kinetic studies of the GdnHCl-induced unfolding of C275/292A-cutinase indicate that this disulfide bond contributes not only to the thermodynamic stability but also to the kinetic stability of LC-cutinase.

Evolution and thermodynamics of the slow unfolding of hyperstable monomeric proteins.

BackgroundThe unfolding speed of some hyperthermophilic proteins is dramatically lower than that of their mesostable homologs. Ribonuclease HII from the hyperthermophilic archaeon Thermococcus kodakaraensis (Tk-RNase HII) is stabilized by its remarkably slow unfolding rate, whereas RNase HI from the thermophilic bacterium Thermus thermophilus (Tt-RNase HI) unfolds rapidly, comparable with to that of RNase HI from Escherichia coli (Ec-RNase HI).ResultsTo clarify whether the difference in the unfolding rate is due to differences in the types of RNase H or differences in proteins from archaea and bacteria, we examined the equilibrium stability and unfolding reaction of RNases HII from the hyperthermophilic bacteria Thermotoga maritima (Tm-RNase HII) and Aquifex aeolicus (Aa-RNase HII) and RNase HI from the hyperthermophilic archaeon Sulfolobus tokodaii (Sto-RNase HI). These proteins from hyperthermophiles are more stable than Ec-RNase HI over all the temperature ranges examined. The observed unfolding speeds of all hyperstable proteins at the different denaturant concentrations studied are much lower than those of Ec-RNase HI, which is in accordance with the familiar slow unfolding of hyperstable proteins. However, the unfolding rate constants of these RNases H in water are dispersed, and the unfolding rate constant of thermophilic archaeal proteins is lower than that of thermophilic bacterial proteins.ConclusionsThese results suggest that the nature of slow unfolding of thermophilic proteins is determined by the evolutionary history of the organisms involved. The unfolding rate constants in water are related to the amount of buried hydrophobic residues in the tertiary structure.

Rational design of a glycosynthase by the crystal structure of β-galactosidase from Bacillus circulans (BgaC) and its use for the synthesis of N-acetyllactosamine type 1 glycan structures

The crystal structure of β-galactosidase from Bacillus circulans (BgaC) was determined at 1.8Å resolution. The overall structure of BgaC consists of three distinct domains, which are the catalytic domain with a TIM-barrel structure and two all-β domains (ABDs). The main-chain fold and steric configurations of the acidic and aromatic residues at the active site were very similar to those of Streptococcus pneumoniae β(1,3)-galactosidase BgaC in complex with galactose. The structure of BgaC was used for the rational design of a glycosynthase. BgaC belongs to the glycoside hydrolase family 35. The essential nucleophilic amino acid residue has been identified as glutamic acid at position 233 by site-directed mutagenesis. Construction of the active site mutant BgaC-Glu233Gly gave rise to a galactosynthase transferring the sugar moiety from α-d-galactopyranosyl fluoride (αGalF) to different β-linked N-acetylglucosamine acceptor substrates in good yield (40-90%) with a remarkably stable product formation. Enzymatic syntheses with BgaC-Glu233Gly afforded the stereo- and regioselective synthesis of β1-3-linked key galactosides like galacto-N-biose or lacto-N-biose.

The N-terminal hybrid binding domain of RNase HI from Thermotoga maritima is important for substrate binding and Mg2+-dependent activity

Thermotoga maritima ribonuclease H (RNase H) I (Tma‐RNase HI) contains a hybrid binding domain (HBD) at the N‐terminal region. To analyze the role of this HBD, Tma‐RNase HI, Tma‐W22A with the single mutation at the HBD, the C‐terminal RNase H domain (Tma‐CD) and the N‐terminal domain containing the HBD (Tma‐ND) were overproduced in Escherichia coli, purified and biochemically characterized. Tma‐RNase HI prefers Mg2+ to Mn2+ for activity, and specifically loses most of the Mg2+‐dependent activity on removal of the HBD and 87% of it by the mutation at the HBD. Tma‐CD lost the ability to suppress the RNase H deficiency of an E. coli rnhA mutant, indicating that the HBD is responsible for in vivo RNase H activity. The cleavage‐site specificities of Tma‐RNase HI are not significantly changed on removal of the HBD, regardless of the metal cofactor. Binding analyses of the proteins to the substrate using surface plasmon resonance indicate that the binding affinity of Tma‐RNase HI is greatly reduced on removal of the HBD or the mutation. These results indicate that there is a correlation between Mg2+‐dependent activity and substrate binding affinity. Tma‐CD was as stable as Tma‐RNase HI, indicating that the HBD is not important for stability. The HBD of Tma‐RNase HI is important not only for substrate binding, but also for Mg2+‐dependent activity, probably because the HBD affects the interaction between the substrate and enzyme at the active site, such that the scissile phosphate group of the substrate and the Mg2+ ion are arranged ideally.

Crystal structure of highly thermostable glycerol kinase from a hyperthermophilic archaeon in a dimeric form.

The crystal structure of glycerol kinase from the hyperthermophilic archaeon Thermococcus kodakaraensis (Tk‐GK) in a dimeric form was determined at a resolution of 2.4 Å. This is the first crystal structure of a hyperthermophilic glycerol kinase. The overall structure of the Tk‐GK dimer is very similar to that of the Escherichia coli glycerol kinase (Ec‐GK) dimer. However, two dimers of Ec‐GK can associate into a tetramer with a twofold axis, whereas those of Tk‐GK cannot. This may be the reason why Tk‐GK is not inhibited by fructose 1,6‐bisphosphate, because the fructose 1,6‐bisphosphate binding site is produced only when a tetrameric structure is formed. Differential scanning calorimetry analyses indicate that Tk‐GK is a highly thermostable protein with a melting temperature (Tm) of 105.4 °C for the major transition. This value is higher than that of Ec‐GK by 34.1 °C. Comparison of the crystal structures of Tk‐GK and Ec‐GK indicate that there is a marked difference in the number of ion pairs in the α16 helix. Four ion pairs, termed IP1–IP4, are formed in this helix in the Tk‐GK structure. To examine whether these ion pairs contribute to the stabilization of Tk‐GK, four Tk‐GK and four Ec‐GK derivatives with reciprocal mutations at the IP1–IP4 sites were constructed. The determination of their stabilities indicates that the removal of each ion pair does not affect the stability of Tk‐GK significantly, whereas the mutations designed to introduce one of these ion pairs stabilize or destabilize Ec‐GK considerably. These results suggest that the ion pairs in the α16 helix contribute to the stabilization of Tk‐GK in a cooperative manner.

A dual role of divalent metal ions in catalysis and folding of RNase H1 from extreme halophilic archaeon Halobacterium sp. NRC-1

RNase H1 from extreme halophilic archaeon Halobacterium sp. NRC‐1 (Halo‐RNH1) consists of an N‐terminal domain with unknown function and a C‐terminal RNase H domain. It is characterized by the high content of acidic residues on the protein surface. The far‐ and near‐UV CD spectra of Halo‐RNH1 suggested that Halo‐RNH1 assumes a partially folded structure in the absence of salt and divalent metal ions. It requires either salt or divalent metal ions for folding. However, thermal denaturation of Halo‐RNH1 analyzed in the presence of salt and/or divalent metal ions by CD spectroscopy suggested that salt and divalent metal ions independently stabilize the protein and thereby facilitate folding. Divalent metal ions stabilize the protein probably by binding mainly to the active site and suppressing negative charge repulsions at this site. Salt stabilizes the protein probably by increasing hydrophobic interactions at the protein core and decreasing negative charge repulsions on the protein surface. Halo‐RNH1 exhibited activity in the presence of divalent metal ions regardless of the presence or absence of 3 M NaCl. However, higher concentrations of divalent metal ions are required for activity in the absence of salt to facilitate folding. Thus, divalent metal ions play a dual role in catalysis and folding of Halo‐RNH1. Construction of the Halo‐RNH1 derivatives lacking an N‐ or C‐terminal domain, followed by biochemical characterizations, indicated that an N‐terminal domain is dispensable for stability, activity, folding, and substrate binding of Halo‐RNH1.

Identification of the substrate binding site in the N-terminal TBP-like domain of RNase H3

Ribonuclease H3 from Bacillus stearothermophilus (Bst‐RNase H3) has the N‐terminal TBP‐like substrate‐binding domain. To identify the substrate binding site in this domain, the mutant proteins of the intact protein and isolated N‐domain, in which six of the seventeen residues corresponding to those involved in DNA binding of TBP are individually mutated to Ala, were constructed. All of them exhibited decreased enzymatic activities and/or substrate‐binding affinities when compared to those of the parent proteins, suggesting that the N‐terminal domain of RNase H3 uses the flat surface of the β‐sheet for substrate binding as TBP to bind DNA. This domain may greatly change conformation upon substrate binding.

Crystal structure of metagenome-derived LC9-RNase H1 with atypical DEDN active site motif

The crystal structure of metagenome‐derived LC9‐RNase H1 was determined. The structure‐based mutational analyses indicated that the active site motif of LC9‐RNase H1 is altered from DEDD to DEDN. In this motif, the location of the second glutamate residue is moved from αA‐helix to β1‐strand immediately next to the first aspartate residue, as in the active site of RNase H2. However, the structure and enzymatic properties of LC9‐RNase H1 highly resemble those of RNase H1, instead of RNase H2. We propose that LC9‐RNase H1 represents bacterial RNases H1 with an atypical DEDN active site motif, which are evolutionarily distinct from those with a typical DEDD active site motif.

Extracellular overproduction and preliminary crystallographic analysis of a family I.3 lipase

A family I.3 lipase from Pseudomonas sp. MIS38 was secreted from Escherichia coli cells to the external medium, purified and crystallized and preliminary crystallographic studies were performed. The crystal was grown at 277 K by the hanging-drop vapour-diffusion method. Native X-ray diffraction data were collected to 1.7 A resolution using synchrotron radiation at station BL38B1, SPring-8. The crystal belongs to space group P2(1), with unit-cell parameters a = 48.79, b = 84.06, c = 87.04 A. Assuming the presence of one molecule per asymmetric unit, the Matthews coefficient V(M) was calculated to be 2.73 A3 Da(-1) and the solvent content was 55%.