Ae Kyung Park

Structural Basis for Antifreeze Activity of Ice-binding Protein from Arctic Yeast

Background: Ice-binding proteins improve the cold tolerance of cells by inhibiting ice growth and recrystallization. Results: Crystal structure and mutagenesis data of LeIBP suggests the B face as an ice-binding site. Conclusion: LeIBP structure adopts a β-helical fold and the aligned Thr/Ser/Ala residues are critical for ice binding. Significance: LeIBP structure can serve as a structural model for a large number of IBPs. Arctic yeast Leucosporidium sp. produces a glycosylated ice-binding protein (LeIBP) with a molecular mass of ∼25 kDa, which can lower the freezing point below the melting point once it binds to ice. LeIBP is a member of a large class of ice-binding proteins, the structures of which are unknown. Here, we report the crystal structures of non-glycosylated LeIBP and glycosylated LeIBP at 1.57- and 2.43-Å resolution, respectively. Structural analysis of the LeIBPs revealed a dimeric right-handed β-helix fold, which is composed of three parts: a large coiled structural domain, a long helix region (residues 96–115 form a long α-helix that packs along one face of the β-helix), and a C-terminal hydrophobic loop region (243PFVPAPEVV251). Unexpectedly, the C-terminal hydrophobic loop region has an extended conformation pointing away from the body of the coiled structural domain and forms intertwined dimer interactions. In addition, structural analysis of glycosylated LeIBP with sugar moieties attached to Asn185 provides a basis for interpreting previous biochemical analyses as well as the increased stability and secretion of glycosylated LeIBP. We also determined that the aligned Thr/Ser/Ala residues are critical for ice binding within the B face of LeIBP using site-directed mutagenesis. Although LeIBP has a common β-helical fold similar to that of canonical hyperactive antifreeze proteins, the ice-binding site is more complex and does not have a simple ice-binding motif. In conclusion, we could identify the ice-binding site of LeIBP and discuss differences in the ice-binding modes compared with other known antifreeze proteins and ice-binding proteins.

Structural understanding of the recycling of oxidized ascorbate by dehydroascorbate reductase (OsDHAR) from Oryza sativa L. japonica

Dehydroascorbate reductase (DHAR) is a key enzyme involved in the recycling of ascorbate, which catalyses the glutathione (GSH)-dependent reduction of oxidized ascorbate (dehydroascorbate, DHA). As a result, DHAR regenerates a pool of reduced ascorbate and detoxifies reactive oxygen species (ROS). In previous experiments involving transgenic rice, we observed that overexpression of DHAR enhanced grain yield and biomass. Since the structure of DHAR is not available, the enzymatic mechanism is not well-understood and remains poorly characterized. To elucidate the molecular basis of DHAR catalysis, we determined the crystal structures of DHAR from Oryza sativa L. japonica (OsDHAR) in the native, ascorbate-bound, and GSH-bound forms and refined their resolutions to 1.9, 1.7, and 1.7 Å, respectively. These complex structures provide the first information regarding the location of the ascorbate and GSH binding sites and their interacting residues. The location of the ascorbate-binding site overlaps with the GSH-binding site, suggesting a ping-pong kinetic mechanism for electron transfer at the common Cys20 active site. Our structural information and mutagenesis data provide useful insights into the reaction mechanism of OsDHAR against ROS-induced oxidative stress in rice.

Crystal structure of the response regulator spr1814 from Streptococcus pneumoniae reveals unique interdomain contacts among NarL family proteins.

Spr1814 belongs to the NarL/FixJ subfamily of signal transduction response regulators (RR), and has been predicted to regulate the neighboring ABC transporter, which translocates antibiotic molecules in Streptococcus pneumoniae. Here, we report the crystal structure of full-length unphosphorylated spr1814 at 1.7Å resolution. The asymmetric unit contains two spr1814 molecules, which display very different conformations. Through comparisons with other RRs structures, we concluded that one molecule adopts a general inactive conformation, whereas the other molecule adopts an intermediate conformation. The superposition of each molecule showed that rotational change of the effector domain occurred in intermediate conformational state, implying that domain rearrangement could occur upon phosphorylation.

Crystal structure of receiver domain of putative NarL family response regulator spr1814 from Streptococcus pneumoniae in the absence and presence of the phosphoryl analog beryllofluoride

Spr1814 of Streptococcus pneumoniae is a putative response regulator (RR) that has four-helix helix-turn-helix DNA-binding domain and belongs to the NarL family. The prototypical RR contains two domains, an N-terminal receiver domain linked to a variable effector domain. The receiver domain functions as a phosphorylation-activated switch and contains the typical doubly wound five-stranded α/β fold. Here, we report the crystal structure of the receiver domain of spr1814 (spr1814(R)) determined in the absence and presence of beryllofluoride as a phosphoryl analog. Based on the overall structure, spr1814(R) was shown to contain the typical fold similar with other structures of the receiver domain; however, an additional linker region connecting the receiver and DNA-binding domain was inserted into the dimer interface of spr1814(R), resulting in the formation of unique dimer interface. Upon phosphorylation, the conformational change of the linker region was observed and this suggests that domain rearrangement between the receiver domain and effector domain could occur in full-length spr1814.

Structure and catalytic mechanism of monodehydroascorbate reductase, MDHAR, from Oryza sativa L. japonica

Ascorbic acid (AsA) maintains redox homeostasis by scavenging reactive oxygen species from prokaryotes to eukaryotes, especially plants. The enzyme monodehydroascorbate reductase (MDHAR) regenerates AsA by catalysing the reduction of monodehydroascorbate, using NADH or NADPH as an electron donor. The detailed recycling mechanism of MDHAR remains unclear due to lack of structural information. Here, we present the crystal structures of MDHAR in the presence of cofactors, nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+), and complexed with AsA as well as its analogue, isoascorbic acid (ISD). The overall structure of MDHAR is similar to other iron-sulphur protein reductases, except for a unique long loop of 63–80 residues, which seems to be essential in forming the active site pocket. From the structural analysis and structure-guided point mutations, we found that the Arg320 residue plays a major substrate binding role, and the Tyr349 residue mediates electron transfer from NAD(P)H to bound substrate via FAD. The enzymatic activity of MDHAR favours NADH as an electron donor over NADPH. Our results show, for the first time, structural insights into this preference. The MDHAR-ISD complex structure revealed an alternative binding conformation of ISD, compared with the MDHAR-AsA complex. This implies a broad substrate (antioxidant) specificity and resulting greater protective ability of MDHAR.

Crystallization and preliminary X-ray crystallographic studies of the ice-binding protein from the Arctic [correction of Aantarctic] yeast Leucosporidium sp. AY30.

Freezing is dangerous to cellular organisms because it causes an increase in the concentration of ions and other solutes in the plasma, denatures biomolecules and ruptures cell membranes. Some cold-adapted organisms can survive at subzero temperatures by producing proteins that bind to and inhibit the growth of ice crystals. To better understand the structure and function of these proteins, the ice-binding protein from Leucosporidium sp. AY30 (LeIBP) was overexpressed, purified and crystallized. The native crystal belonged to space group P4(3)2(1)2, with unit-cell parameters a=b=98.05, c=106.13 Å. Since LeIBP lacks any cysteine or methionine residues, two leucine residues (Leu69 and Leu155) were substituted by methionine residues in order to obtain selenomethionine-substituted LeIBP for use in multiple-wavelength anomalous diffraction (MAD) phasing. The selenomethionine-substituted mutant crystallized in the same space group as the native protein.

Crystallization and preliminary X‐ray crystallographic studies of the ice‐binding protein from the Antarctic yeast Leucosporidium sp. AY30. Corrigendum

A correction to the article by Park et al. [(2011). Acta Cryst. F67, 800–802].

Potential Application of the Oryza sativa Monodehydroascorbate Reductase Gene (OsMDHAR) to Improve the Stress Tolerance and Fermentative Capacity of Saccharomyces cerevisiae

Monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) is an important enzyme for ascorbate recycling. To examine whether heterologous expression of MDHAR from Oryza sativa (OsMDHAR) can prevent the deleterious effects of unfavorable growth conditions, we constructed a transgenic yeast strain harboring a recombinant plasmid carrying OsMDHAR (p426GPD::OsMDHAR). OsMDHAR-expressing yeast cells displayed enhanced tolerance to hydrogen peroxide by maintaining redox homoeostasis, proteostasis, and the ascorbate (AsA)-like pool following the accumulation of antioxidant enzymes and molecules, metabolic enzymes, and molecular chaperones and their cofactors, compared to wild-type (WT) cells carrying vector alone. The addition of exogenous AsA or its analogue isoascorbic acid increased the viability of WT and ara2Δ cells under oxidative stress. Furthermore, the survival of OsMDHAR-expressing cells was greater than that of WT cells when cells at mid-log growth phase were exposed to high concentrations of ethanol. High OsMDHAR expression also improved the fermentative capacity of the yeast during glucose-based batch fermentation at a standard cultivation temperature (30°C). The alcohol yield of OsMDHAR-expressing transgenic yeast during fermentation was approximately 25% (0.18 g·g-1) higher than that of WT yeast. Accordingly, OsMDHAR-expressing transgenic yeast showed prolonged survival during the environmental stresses produced during fermentation. These results suggest that heterologous OsMDHAR expression increases tolerance to reactive oxygen species-induced oxidative stress by improving cellular redox homeostasis and improves survival during fermentation, which enhances fermentative capacity.

The structure of a shellfish specific GST class glutathione S‐transferase from antarctic bivalve Laternula elliptica reveals novel active site architecture

Glutathione‐S‐transferases have been identified in all the living species examined so far, yet little is known about their function in marine organisms. In a previous report, the recently identified GST from Antarctic bivalve Laternula elliptica (LeGST) was classified into the rho class GST, but there are several unique features of LeGST that may justify reclassification, which could represent specific shellfish GSTs. Here, we determined the crystal structure of LeGST, which is a shellfish specific class of GST. The structural analysis showed that the relatively open and wide hydrophobic H‐site of the LeGST allows this GST to accommodate various substrates. These results suggest that the H‐site of LeGST may be the result of adaptation to their environments as sedentary organisms. Proteins 2013.

Expression, purification and preliminary X-ray crystallographic analysis of nitroalkane oxidase (NAO) from Pseudomonas aeruginosa

Nitroalkane oxidase (NAO) is a flavin-dependent enzyme which catalyses the oxidation of nitroalkanes to the corresponding aldehydes or ketones, nitrite and hydrogen peroxide. In order to better understand the structure and function of this enzyme, NAO from Pseudomonas aeruginosa was purified and crystallized as a native and a selenomethionine-substituted (SeMet) enzyme. Both crystals diffracted to a resolution of 1.9 Å and belonged to the primitive orthorhombic space group P2₁, with unit-cell parameters a = 70.06, b = 55.43, c = 87.74 Å, β = 96.56° for native NAO and a = 69.89, b = 54.83, c = 88.20 Å, β = 95.79° for SeMet NAO. Assuming the presence of two molecules in the asymmetric unit in both crystals, the Matthews coefficients (VM) for native and SeMet NAO were calculated to be 2.30 and 2.48 ų Da⁻¹, with estimated solvent contents of 46.50 and 50.37%, respectively.