Amit Priyadarshi

Structural insights into Staphylococcus aureus enoyl-ACP reductase (FabI), in complex with NADP and triclosan.

Structural insights into Staphylococcus aureus enoyl-ACP reductase (FabI), in complex with NADP and triclosan Amit Priyadarshi, Eunice EunKyeong Kim,* and Kwang Yeon Hwang* 1Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-701, South Korea 2 Biomedical Research Center, Korea Institute of Science and Technology, Seoul 136-791, South Korea 3 Biomolecular Science, University of Science and Technology, Yuseong-gu, Daejeon 305-333, South Korea

Structural and functional analysis of a novel EstE5 belonging to the subfamily of hormone-sensitive lipase.

Hormone-sensitive lipase (HSL) plays an important role in the regulation of rodent fat cell lipolysis. It is regarded as an adipose tissue-specific enzyme whose sole metabolic role is the catalysis of hormone-stimulated lipolysis in mammalian cells. In this report we describe the functional and structural analysis of an EstE5 protein from a soil metagenome library. Function analysis results indicated that EstE5 preferentially hydrolyzes short-chain ester compounds, and our kinetic studies revealed the optimal pH and temperature. Based on the structural analysis, we defined the active site and the binding pocket. Structurally, EstE5 belongs to the HSL family and these structural studies may have applications in the production of value-added products, including pharmaceuticals.

Structural and functional analysis of a novel hormone-sensitive lipase from a metagenome library

Structural and functional analysis of a novel hormone-sensitive lipase from a metagenome library Ki Hyun Nam,1y Min-Young Kim,2y Soo-Jin Kim, Amit Priyadarshi, Suk-Tae Kwon, Bon-Sung Koo, Sang-Hong Yoon, and Kwang Yeon Hwang* 1Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea 2National Agrobiodiversity Center, National Academy of Agricultural Science, Rural Development Administration, Suwon 441-707, Korea 3 Biomedical Research Center, Korea Institute of Science and Technology, Seoul 136-791, Korea 4Department of Genetic Engineering, Sungkyunkwan University, Jangan-gu, Suwon 440-746, Korea 5 Fermentation and Food Processing Division, Department of Korean Food Research for Globalization, National Academy of Agricultural Science,

The crystal structure of an HSL-homolog EstE5 complex with PMSF reveals a unique configuration that inhibits the nucleophile Ser144 in catalytic triads.

The esterase/lipase family (EC 3.1.1.3/EC 3.1.1.1) represents a diverse group of hydrolases that catalyze the cleavage of ester bonds and are widely distributed in animals, plants and microorganisms. Among these enzymes, hormone-sensitive lipases, play a critical role in the regulation of rodent fat cell lipolysis and are regarded as adipose tissue-specific enzymes. Recently, we reported the structural and biological characterization of EstE5 from the metagenome library [K.H. Nam, M.Y. Kim, S.J. Kim, A. Priyadarshi, W.H. Lee, K.Y. Hwang, Structural and functional analysis of a novel EstE5 belonging to the subfamily of hormone-sensitive lipase, Biochem. Biophys. Res. Commun. 379 (2009) 553-556]. The structure of this protein revealed that it belongs to the HSL-family. Here, we report the inhibition of the activity of the HSL-homolog EstE5 protein as determined by the use of esterase/lipase inhibitors. Our results revealed that the EstE5 protein is significantly inhibited by PMSF. In addition, this is the first study to identify the crystal structures of EstE5-PMSF at 2.4 and 2.5A among the HSL-homolog structures. This structural configuration is similar to that adopted when serine proteases are inhibited by PMSF. The results presented here provide valuable information regarding the properties of the HSL-family.

Structural insights into mouse anti-apoptotic Bcl-xl reveal affinity for Beclin 1 and gossypol.

This study reports the crystal structures of Bcl-xl wild type and three Bcl-xl mutants (Y101A, F105A, and R139A) with amino acid substitutions in the hydrophobic groove of the Bcl-xl BH3 domain. An additional 12 ordered residues were observed in a highly flexible loop between the alpha1 and alpha2 helices, and were recognized as an important deamidation site for the regulation of apoptosis. The autophagy-effector protein, Beclin 1, contains a novel BH3 domain (residues 101-125), which binds to the surface cleft of Bcl-xl, as confirmed by nuclear magnetic resonance (NMR) spectroscopy and analytical gel-filtration results. Gossypol, a potent inhibitor of Bcl-xl, had a K(d) value of 0.9 microM. In addition, the structural and biochemical analysis of five Bcl-xl substitution mutants will provide structural insights into the design and development of anti-cancer drugs.

Structural and functional analysis of Vitamin K2 synthesis protein MenD

Here we describe in detail the crystal structures of the Vitamin K(2) synthesis protein MenD, from Escherichia coli, in complex with thiamine diphosphate (ThDP) and oxoglutarate, and the effects of cofactor and substrate on its structural stability. This is the first reported structure of MenD in complex with oxoglutarate. The residues Gly472 to Phe488 of the active site region are either disordered, or in an open conformation in the MenD oxoglutarate complex structure, but adopt a closed conformation in the MenD ThDP complex structure. Biospecific-interaction analysis using surface plasmon resonance (SPR) technology reveals an affinity for ThDP and oxoglutarate in the nanomolar range. Biochemical and structural analysis confirmed that MenD is highly dependent on ThDP for its structural stability. Our structural results combined with the biochemical assay reveal novel features of the enzyme that could be utilized in a program of rational structure-based drug design, as well as in helping to enhance our knowledge of the menaquinone synthesis pathway in greater detail.

Structural insights of the MenD from Escherichia coli reveal ThDP affinity

MenD (2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate) synthase belongs to the superfamily of thiamin diphosphate-dependent decarboxylases, which converts isochorismate and 2-oxoglutarate to SHCHC, pyruvate, and carbon dioxide. Here, we report the first crystal structure of apo-MenD from Escherichia coli determined in tetragonal crystal form. The subunit displays the typical three-domain structure observed for ThDP-dependent enzymes. Analytical gel filtration shows that EcMenD behaves as a dimer as well as a tetramer. Circular dichroism and isothermal calorimetry results confirm EcMenD dependency on ThDP, which concomitantly helps to stabilize with better configuration.

Crystal structure of bacterioferritin from Rhodobacter sphaeroides

Iron is essential for the survival of organisms, but either excess or deficient levels of iron induce oxidative stress, thereby causing cell damage. As a result, iron regulation is essential for proper cell growth and proliferation in most organisms. Bacterioferritin is a ferritin-like family protein that contains a heme molecule and a ferroxidase site at the di-iron center. This protein plays a primary role in intracellular iron storage for iron homeostasis, as well as in the maintenance of iron in a soluble and non-toxic form. Although several bacterioferritin structures have been determined, no structural studies have successfully elucidated the molecular function of the heme molecule and the ferroxidase center. Here, we report the crystal structure of bacterioferritin from Rhodobacter sphaeroides. This protein exists in a roughly spherical configuration via the assembly of 24 subunits. We describe the oligomeric arrangement, ferroxidase center and heme-binding site based on this structure. The protein contains a single iron-binding configuration in the ferroxidase center, which allows for the release of iron by His130 when the protein is in the intermediate state. The heme molecule in RsBfr is stabilized by shifting of the van der Waals interaction center between the porphyrin of the heme and Trp26. We anticipate that further structural analysis will provide a more complete understanding of the molecular mechanisms of members of the ferritin-like family.

Crystal structure of the tRNA‐specific adenosine deaminase from Streptococcus pyogenes

Crystal structure of the tRNA-specific adenosine deaminase from Streptococcus pyogenes Won-Ho Lee, Young Kwan Kim, Ki Hyun Nam, Amit Priyadarshi, Eun Hye Lee, Eunice Eunkyeong Kim, Young Ho Jeon, Chaejoon Cheong, and Kwang Yeon Hwang* 1 Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-701, South Korea 2 Biomedical Research Center, Korea Institute of Science and Technology, Seoul 136-791, South Korea 3 Magnetic Resonance Team, Korea Basic Science Institute, Ochang, Chungbuk 363-883, Korea

Insight into the antibacterial drug design and architectural mechanism of peptide recognition from the E. faecium peptide deformylase structure

Insight into the antibacterial drug design and architectural mechanism of peptide recognition from the E. faecium peptide deformylase structure Ki Hyun Nam,1y Jung Il Ham,1,2y Amit Priyadarshi, Eunice Eunkyeong Kim, NamHyun Chung, and Kwang Yeon Hwang* 1Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-713, Korea 2Department of Medicine Laboratory, National Cancer Center, Gyeounggi 410-769, Korea 3 Biomedical Research Center, Korea Institute of Science and Technology, Seoul 136-791, Korea