Exploring the smallest active fragment of HsQSOX1b and finding a highly efficient oxidative engine.
Human quiescin-sulfhydryl oxidase 1 isoform b (HsQSOX1b) is a highly efficient, multiple-domain enzyme that directly inserts disulfide bonds into client protein. However, previous studies have focused mainly on the catalytic activity of the whole protein rather than its domain structure. In this research, we dissected the structure and function of HsQSOX1b and explored its mechanism as a highly efficient sulfhydryl oxidase by analyzing the truncated variants. The results showed that the first HsQSOX1b thioredoxin domain was essential for thiol oxidase activity. The smallest active fragment (SAQ) was identified to consist of a helix-rich region (HRR) and an essential for respiration and viability/augmenter of liver regeneration (ERV/ALR) domain, which remained highly active to oxidize an artificial non-thiol substrate but not small molecular and protein thiols. Our study clearly demonstrated that SAQ is a highly efficient oxidative engine, which shows high efficiency in the de novo disulfide formation and oxygen reduction and that this more efficient oxidative engine is necessary for the highly efficient catalysis of QSOXs compared to Erv1 and Erv2. This study will help address the roles of different HsQSOX1b domains in de novo disulfide formation and encourage the engineering of more efficient QSOX variants for the in vitro folding of disulfide-containing proteins.
Synthetic Biology and Biotechnology Laboratory, School of Pharmacy, East China University of Science and Technology, Shanghai, China.
Wenyun
W
Zheng
Wenyao
W
Zhang
Wei
W
Hu
Chao
C
Zhang
Yi
Y
Yang
D002384:Catalysis; D006801:Humans; D007527:Isoenzymes; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000737:chemistry* / Q000378:metabolism*; D010720:Phosphines / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs; D013329:Structure-Activity Relationship; D013879:Thioredoxins / Q000737:chemistry / Q000378:metabolism
D007527:Isoenzymes; D010088:Oxidoreductases; D050862:Oxidoreductases Acting on Sulfur Group Donors; D010720:Phosphines; D013879:Thioredoxins; C080938:tris(2-carboxyethyl)phosphine; C112161:thiol oxidase, 47 kDa; C117183:QSOX1 protein, human