Exploring the Smallest Active Fragment of HsQSOX1b and Finding a Highly Efficient Oxidative Engine

Wenyun Zheng,Yi Yang,Wenyao Zhang,Chao Zhang,Wei Hu,Luis Eduardo Soares Netto

doi:10.1371/journal.pone.0040935

Wenyun Zheng, Yi Yang + Show 4 more

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https://doi.org/10.1371/journal.pone.0040935

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Journal: PloS one	Publication Date: Jul 20, 2012
Citations: 13	License type: CC BY 4.0

Affiliation: East China University of Science and Technology

Abstract

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.

Highlights

Quiescin-sulfhydryl oxidase are present in most eukaryotes [1,2,3] but not in yeast/fungi [4]
We found that the variants without two Trx domains showed the similar tris [2-carboxyethyl] phosphine (TCEP) oxidase activity to the full-length enzyme, indicating that those two domains did not affect the folding of the other part of HsQSOX1b (Fig. 1B)
The kcat values of the variants HsQSOX1b267–604 and HsQSOX1b295–604 were higher than that of SAQ and much higher than that of the Erv1 or Erv2. These results suggest that SAQ, acted as the oxidative engine, show high efficiency in the de novo disulfide formation and oxygen reduction, including the reduction of the C449–C452 disulfide and the electron transfer from this active site to flavin adenine dinucleotide (FAD) and to oxygen

Summary

Introduction

Quiescin-sulfhydryl oxidase are present in most eukaryotes [1,2,3] but not in yeast/fungi [4]. QSOXs belong to a group of enzymes called sulfhydryl oxidases, which includes the flavin adenine dinucleotide (FAD) prosthetic group, and oxidize small molecular thiolcontaining substrate, i.g. dithiothreitol (DTT) and glutathione (GSH) [1,11] Among these enzymes, only QSOXs are capable of the facile and direct insertion of disulfide bonds into reduced client proteins, which is termed protein thiol oxidase activity. TCEP has been widely used for reduction of disulfide containing proteins and directly reduces the de novo formed disulfides residing in the active sites of these enzymes, regenerates free thiols, and makes the enzyme cycling and generating hydrogen peroxide from oxygen [12,13]. Comparing to endoplasmic reticulum oxidoreductin 1 (Ero1) and Erv family of sulfhydryl oxidases, QSOXs have shown hundredfold higher thiol oxidase and TCEP oxidase activities compared with the Ero and Erv families of sulfhydryl oxidases [10,12]

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