Han-Woo Kim

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.

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.

Characterization of Two Antimicrobial Peptides from Antarctic Fishes (Notothenia coriiceps and Parachaenichthys charcoti)

We identified two antimicrobial peptides (AMPs) with similarity to moronecidin in Antarctic fishes. The characteristics of both AMPs were determined using moronecidin as a control. Moronecidin, which was first isolated from hybrid striped bass, is highly salt-resistant, and possesses broad-spectrum activity against various microbes. The moronecidin-like peptide from Notothenia coriiceps exhibited a narrower spectrum of activity and a higher salt sensitivity than moronecidin. The AMP from Parachaenichthys charcoti exhibited similar antimicrobial activity to moronecidin, and similar salt sensitivity. In an experiment to identify toxic effects, both of the moronecidin-like peptides from the Antarctic fishes exhibited lower hemolytic activity than moronecidin. In spite of its low toxicity, the AMP from N. coriiceps is unlikely to be considered as a candidate for antibiotic development, owing to its narrow spectrum of activity and high salt sensitivity. In contrast, the high salt resistance and broad-spectrum activity of the AMP from P. charcoti could be more advantageous for clinical use than moronecidin, since it could kill bacteria under physiological conditions with low toxicity. A further comparison of these two AMPs from Antarctic fishes with other AMPs could help to reduce the toxicity of AMPs for clinical use.

Purification, characterization and preliminary X-ray crystallographic studies of monodehydroascorbate reductase from Oryza sativa L. japonica

Monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) is a key enzyme in the reactive oxygen species (ROS) detoxification system of plants. The participation of MDHAR in ascorbate (AsA) recycling in the ascorbate-glutathione cycle is important in the acquired tolerance of crop plants to abiotic environmental stresses. Thus, MDHAR represents a strategic target protein for the improvement of crop yields. Although physiological studies have intensively characterized MDHAR, a structure-based functional analysis is not available. Here, a cytosolic MDHAR (OsMDHAR) derived from Oryza sativa L. japonica was expressed using Escherichia coli strain NiCo21 (DE3) and purified. The purified OsMDHAR showed specific enzyme activity (approximately 380 U per milligram of protein) and was crystallized using the hanging-drop vapour-diffusion method at pH 8.0 and 298 K. The crystal diffracted to 1.9 Å resolution and contained one molecule in the asymmetric unit (the Matthews coefficient VM is 1.98 Å(3) Da(-1), corresponding to a solvent content of 38.06%) in space group P41212 with unit-cell parameters a = b = 81.89, c = 120.4 Å. The phase of the OsMDHAR structure was resolved by the molecular-replacement method using a ferredoxin reductase from Acidovorax sp. strain KKS102 (PDB entry 4h4q) as a model.

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.

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.

Identification of proteolytic bacteria from the Arctic Chukchi Sea expedition cruise and characterization of cold-active proteases

Following collection of seawater samples during an Arctic Chukchi Sea expedition cruise of the Korean icebreaker Araon in 2012, a total of 15,696 bacteria were randomly isolated from Marine Broth 2216 agar plates. Of these, 2,526 (16%) showed proteolytic activity and were identified as mainly Alteromonas (31%), Staphylococcus (27%), and Pseudoalteromonas (14%). Among the proteolytic strains, seven were selected based on their significant ability to grow and produce a halo on skim milk plates at low temperatures (<5°C) owing to cold-active proteases. These strains were affiliated with the genus Pseudoalteromonas and were divided into three groups based on phylogenetic analysis of the 16S rRNA genes. Profiling cell membrane fatty acids confirmed the 16S rRNA-based differentiation and revealed the accordance between the two analyses. Seven genes for serine protease precursors were amplified from the corresponding strains, and based on sequence similarities, these genes were divided into three groups that were identical to those identified by the 16S rRNA phylogenetic analysis. Three protease genes from the representative strains of each group were composed of 2,127–2,130 bp, encoding 708–709 amino acids, and these genes yielded products with calculated molecular weights of approximately 72.3–72.8 kDa. Amino acid sequence analysis suggested that the precursors are members of the subtilase serine endo- and exo-peptidase clan and contain four domains (signal peptide, N-terminal prosequence, catalytic domain, and two pre-peptidase C-terminal domains). Upon expression in E. coli, each recombinant protease exhibited proteolytic activity on zymogram gels.

Enhancing Extracellular Lipolytic Enzyme Production In An Arctic Bacterium, Psychrobacter sp. ArcL13, By Using Statistical Optimization And Fed-Batch Fermentation

A strain isolated from seawater samples in the Chuckchi Sea and exhibiting extracellular lipolytic activity was identified using 16S rRNA gene sequence analysis as Psychrobacter sp. ArcL13. The lipolytic enzyme exhibited cold-active properties and high hydrolytic activity toward p-nitrophenyl caprylate (C8), p-nitrophenyl decanoate (C10), and sunflower oil. Statistical optimization of the medium components was performed to enhance the production of cold-active extracellular lipolytic activity. Glucose, yeast extract (YE), and NaCl were selected as the main efficient nutrient sources. Fed-batch fermentation using optimized medium with concentrated YE as the main feeding material showed a maximum lipolytic activity of 10.7 U/mL, which was a 21-fold increase in production over unoptimized flask culture conditions. The information obtained in the present study could prove applicable to the production of cold-active lipase on a large scale.

Crystal structure of the inactive state of the receiver domain of Spo0A from Paenisporosarcina sp. TG-14, a psychrophilic bacterium isolated from an Antarctic glacier

The two-component phosphorelay system is the most prevalent mechanism for sensing and transducing environmental signals in bacteria. Spore formation, which relies on the two-component phosphorelay system, enables the long-term survival of the glacial bacterium Paenisporosarcina sp. TG-14 in the extreme cold environment. Spo0A is a key response regulator of the phosphorelay system in the early stage of spore formation. The protein is composed of a regulatory N-terminal phospho-receiver domain and a DNA-binding C-terminal activator domain. We solved the three-dimensional structure of the unphosphorylated (inactive) form of the receiver domain of Spo0A (PaSpo0A-R) from Paenisporosarcina sp. TG-14. A structural comparison with phosphorylated (active form) Spo0A from Bacillus stearothermophilus (BsSpo0A) showed minor notable differences. A molecular dynamics study of a model of the active form and the crystal structures revealed significant differences in the α4 helix and the preceding loop region where phosphorylation occurs. Although an oligomerization study of PaSpo0A-R by analytical ultracentrifugation (AUC) has shown that the protein is in a monomeric state in solution, both crosslinking and crystal-packing analyses indicate the possibility of weak dimer formation by a previously undocumented mechanism. Collectively, these observations provide insight into the mechanism of phosphorylation-dependent activation unique to Spo0A.

Complete genome sequence of the crude oil-degrading thermophilic bacterium Geobacillus sp. JS12.

Here, we report the complete genome sequence of Geobacillus sp. JS12, isolated from composts located in Namhae, Korea, which shows extracellular lipolytic activities at high temperatures. An array of genes related to the utilization of lipids was identified by whole genome analysis. The genome sequence of the strain JS12 provides basic information for wider exploitation of thermostable industrial lipases.