Sulfonucleotide reductase in complexes with substrate and product reveals the structural snapshots in the catalytic cycle

• Main Authors: I-Shao Chen, 陳奕劭
• Other Authors: Chun-Hua,Hsu
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/05289456503473097533

碩士 === 國立臺灣大學 === 農業化學研究所 === 99 === The first committed step of reductive sulfate assimilation in the biosynthesis of cysteine in pathogenic bacteria is catalyzed by sulfonucleotide reductases (SNRases). However, mammals do not possess the sulfate reduction pathway, which makes sulfonucleotide reductases being a promising target for drug development against human pathogens. To elucidate the catalytic mechanism, ssSNRase (sulfonucleotide reductase from Sulfolobus solfataricus) was studied by a combination of biochemical, spectroscopic, and crystallographic approaches. Purified ssSNRase protein in solution is shown brownish in color and proposed it should contain one [4Fe-4S] cluster per polypeptide chain. Data from ultraviolet-visible absorption spectroscopy and X-ray fluorescence scan were collected to elucidate the nature of the prosthetic group containing property. CD experiments showed that ssSNRase possesses high melting temperature around 82°C, which consistent with the physiological growth environment of Sulfolobus solfataricus. Crystal structure of ssSNRases in complex with its product AMP and its substrate APS were solved to 1.97 and 2.03 Å, respectively. Residues that are essential for catalytic activity could be elucidated by comparing substrate-product substituted active sites and the possible function of these residues were also proposed. C-terminus tail containing active Cys231 residue in an organized H-bonding network with the channel of enzyme were both observed. By these two snapshots of ssSNRase catalytic states, we can provide more information about the C-terminus tail displacement and the mobility model of the C-terminal tail during APS reduction can be modified.