https://repository.kopri.re.kr/handle/201206/9726 2026-03-05T10:03:40Z https://repository.kopri.re.kr/handle/201206/10840 Title: Characterization and Structural Determination of Cold-Adapted Monodehydroascorbate Reductase, MDHAR, from the Antarctic Hairgrass Deschampsia Antarctica Authors: Park, Ae Kyung; Kim, Il-Sup; Do, Hackwon; Kim, Hyun; Choi, Woong; Jo, Seung-Woo; Shin, Seung Chul; Lee, Jun Hyuck; Yoon, Ho-Sung; Kim, Han-Woo Abstract: Ascorbic acid (AsA) is an abundant component of plants and acts as a strong and active antioxidant. In order to maintain the antioxidative capacity of AsA, the rapid regeneration of AsA is regulated by dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR). To understand how MDHAR functions under extreme temperature conditions, this study characterized its biochemical properties and determined the crystal structure of MDHAR from the Antarctic hairgrass Deschampsia antarctica (DaMDHAR) at 2.2 A resolution. This allowed for a structural comparison with the mesophilic MDHAR from Oryza sativa L. japonica (OsMDHAR). In the functional analysis, yeast cells expressing DaMDHAR were tolerant to freezing and thawing cycles. It is possible that the expression of DaMDHAR in yeast enhanced the tolerance for ROS-induced abiotic stress 2019-10-01T00:00:00Z https://repository.kopri.re.kr/handle/201206/10939 Title: Structural and functional characterization of a novel cold-active S-formylglutathione hydrolase (SfSFGH) homolog from Shewanella frigidimarina, a psychrophilic bacterium Authors: Lee, Chang Woo; Yoo, Wanki; Park, Sun-Ha; Lee, Ly Thi Huong Luu; Jeong, Chang？Sook; Ryu, Bum Han; Shin, Seung Chul; Kim, Han-Woo; Park, Hyun; Kim, Kyeong Kyu; Kim, T. Doohun; Lee, Jun Hyuck Abstract: Background: S-formylglutathione is hydrolyzed to glutathione and formate by an S-formylglutathione hydrolase (SFGH) (3.1.2.12). This thiol esterase belongs to the esterase family and is also known as esterase D. SFGHs contain highly conserved active residues of Ser-Asp-His as a catalytic triad at the active site. Characterization and investigation of SFGH from Antarctic organisms at the molecular level is needed for industrial use through protein engineering. Results: A novel cold-active S-formylglutathione hydrolase (SfSFGH) from Shewanella frigidimarina, composed of 279 amino acids with a molecular mass of ~31.0 kDa, was characterized. Sequence analysis of SfSFGH revealed a conserved pentapeptide of G-X-S-X-G found in various lipolytic enzymes along with a putative catalytic triad of Ser148-Asp224-His257. Activity analysis showed that SfSFGH was active towards short-chain esters, such as p-nitrophenyl acetate, butyrate, hexanoate, and octanoate. The optimum pH for enzymatic activity was slightly alkaline (pH 8.0). To investigate the active site configuration of SfSFGH, we determined the crystal structure of SfSFGH at 2.32 A resolution. Structural analysis shows that a Trp182 residue is located at the active site entrance, allowing it to act as a gatekeeper residue to control substrate binding to SfSFGH. Moreover, SfSFGH displayed more than 50% of its initial activity in the presence of various chemicals, including 30% EtOH, 1% Triton X-100, 1% SDS, and 5 M urea. Conclusions: Mutation of Trp182 to Ala allowed SfSFGH to accommodate a longer chain of substrates. It is thought that the W182A mutation increases the substrate-binding pocket and decreases the steric effect for larger substrates in SfSFGH. Consequently, the W182A mutant has a broader substrate specificity compared to wild-type SfSFGH. Taken together, this study provides useful structure-function data of a SFGH family member and may inform protein engineering strategies for industrial applications of SfSFGH. 2019-08-01T00:00:00Z https://repository.kopri.re.kr/handle/201206/10891 Title: Nanopore sequencing reads improve assembly and gene annotation of the Parochlus steinenii genome Authors: Shin, Seung Chul; Kim, Hyun; Lee, Jun Hyuck; Kim, Han-Woo; Park, Joonho; Choi, Beom-Soon; Lee, Sang-Choon; Kim, Ji Hee; Lee, Hyoungseok; Kim, Sanghee Abstract: Parochlus steinenii is a winged midge from King George Island. It is cold-tolerant and endures the harsh Antarctic winter. Previously, we reported the genome of this midge, but the genome assembly with short reads had limited contig contiguity, which reduced the completeness of the genome assembly and the annotated gene sets. Recently, assembly contiguity has been increased using nanopore technology. A number of methods for enhancing the low base quality of the assembly have been reported, including long-read (e.g. Nanopolish) or short-read (e.g. Pilon) based methods. Based on these advances, we used nanopore technologies to upgrade the draft genome sequence of P. steinenii. The final assembled genome was 145,366,448 bases in length. The contig number decreased from 9,132 to 162, and the N50 contig size increased from 36,946 to 1,989,550 bases. The BUSCO completeness of the assembly increased from 87.8 to 98.7%. Improved assembly statistics helped predict more genes from the draft genome of P. steinenii. The completeness of the predicted gene model increased from 79.5 to 92.1%, but the numbers and types of the predicted repeats were similar to those observed in the short read assembly, with the exception of long interspersed nuclear elements. In the present study, we markedly improved the P. steinenii genome assembly statistics using nanopore sequencing, but found that genome polishing with high-quality reads was essential for improving genome annotation. The number of genes predicted and the lengths of the genes were greater than before, and nanopore technology readily improved genome information. 2019-03-01T00:00:00Z