大肠杆菌二硫键形成蛋白A（DsbA）研究进展

二硫键形成蛋白A(DisulfidebondformationproteinA,DsbA)是存在于大肠杆菌周质胞腔内的一种参与新生蛋白质折叠过程中催化二硫键形成的折叠酶。综述了DsbA三维结构、进化过程、协助蛋白质体内外复性方面的研究进展。DsbA比硫氧还原蛋白具有更强的氧化性,其强氧化性来自于Cys30残基异常低的pKa值和不稳定的氧化型结构,通过定点突变的研究表明了Cys30残基是DsbA活性中心最关键的氨基酸残基之一。DsbA不论在体内与目标蛋白融合表达还是在体外以折叠酶形式添加,都能有效地催化蛋白质的折叠复性,同时DsbA还具有部分分子伴侣的活性。     

色氨酸类似物标记法研究DsbA蛋白的结构环境

用生物标记的方法将色氨酸类似物标记在DsbA蛋白中的色氨酸位置,分析标记蛋白质的谱学性质、色氨酸结构环境和潜在应用前景.5-OH-Trp标记的DsbA蛋白具有315 nm激发的荧光发射光谱;¹⁹F-NMR 能分辨5-F-Trp标记的DsbA蛋白的两个F-Trp残基(Trp76和Trp126),Trp76化学位移变化反映二硫键交换引起的结构转化.进一步将利用标记蛋白的独特荧光和¹⁹F-NMR性质,研究DsbA蛋白的氧化还原及与底物蛋白的结合作用.     

二硫键异构酶的纯化及其对重组蛋白体外折叠的影响

自牛肝中纯化了蛋白二硫键异构酶(PDI),并对重组蛋白的酶促折叠过程进行了探讨．结果表明。在等摩尔PDl的催化作用下,可使1mg／ml的IIJ-2的正确折叠率提高到58%以上,比活性由4×10⁶u／mg增加到8．2×10⁶u／mg,PDl还能部分纠正二硫键错配的IL-2异构体成为正确折叠的lL-2和防止IL-2通过cys的链问交联形成聚合体。GM-CSF在PDl催化下也有类似的结果。PDl作用的关键是它所催化的琉基一二硫键的交换反应。     

蛋白质的氧化重折叠

经过近几十年来广泛而深入的研究,蛋白质氧化重折叠的机制已得到相当详细的阐明。1在已研究过的蛋白质中,大多数蛋白质都是沿着多途径而非单一、特定的途径进行氧化重折叠,这与折叠能量景观学说是一致的。2正是氨基酸残基间的天然相互作用而不是非天然的相互作用控制蛋白质的折叠过程。这一结论与含非天然二硫键的折叠中间体在牛胰蛋白酶抑制剂（BPTI）折叠中所起的重要作用并非相互排斥,因为后者仅仅是进行链内二硫键重排的化学反应所必需,与控制肽链折叠无直接关系。3根据对BPTI的研究,二硫键曾被认为仅仅具有稳定蛋白质天然结构的作用,既不决定折叠途径也不决定其三维构象。这一观点不适用于其它蛋白质。对凝乳酶原的研究表明,天然二硫键的形成是恢复天然构象的前提。天然二硫键的形成与肽键的正确折叠相辅相成,更具有普遍意义。4在氧化重折叠的早期,二硫键的形成基本上是一个随机过程,随着肽链的折叠二硫键的形成越来越受折叠中间体构象的限制。提高重组蛋白质的复性产率是生物技术领域中的一个巨大的挑战。除了分子聚集外,在折叠过程中所形成的二硫键错配分子是导致低复性率的另一个主要原因。氧化重折叠机制的阐明为解决此问题提供了有益的启示。如上所述,在折叠的后期,二硫键的形成决定于折叠中间体的构象,类天然、有柔性的结构有利于天然二硫键形成和正确折叠,具有这类结构的分子为有效的折叠中间体,最终都能转变为天然产物;而无效折叠中间体往往具有稳定的结构,使巯基、二硫键内埋妨碍二硫键重排,并因能垒的障碍不利于进一步折叠。因此,降低无效折叠中间体的稳定性使之转变为有效折叠中间体是提高含二硫键蛋白质复性率的一条基本原则,实验证明,碱性pH、低温、降低蛋白质稳定性的试剂、蛋白质二硫键异构酶、改变蛋白质一级结构是实现这一原则的有效手段。此外,这里还就氧化重折叠的基础和应用研究的前景进行了讨论。     

分子伴侣 SecB 基因和人淋巴毒素基因在大肠杆菌中的共表达

分于伴侣(Chaperohe)是细胞内催化及维持其他蛋白质正确梅象的一类蛋白质分子^[1,2]。研究表明,分子伴侣参与细胞内许多蛋白质的折叠、聚合以及跨膜运输^[3,4],通过瞬时稳定其他蛋白质折叠中间体,阻止了蛋白中间体的聚集,帮助其形成正确构象^[5,6]。SecB是一个胞质酸性蛋白．单体分子量为17kDa,在体内以4～6个相同亚基组成的寡聚体形式存在。它在大肠杆菌中参与蛋白质分泌系统,纯化后进行离体试验表明,它可以阻止抗蛋白酶的pre-MBP的出现,能稳定地结合前体蛋白．使其处于适合运输的构型^[7],它的作用是使蛋白质可以在正确折叠前跨过细胞膜,运输到细胞周质中。SecB通过与前体蛋白结合．从而阻止前体蛋自由于不正确折叠发生的聚集,属于分子伴侣家族的成员。分子伴侣的这些特性使得它们在基因工程中具有广阔的应用前景。外源蛋白在大肠杆菌中高表达时往往形成无活性的包涵体,包涵体大多是蛋白质在过量表达过程中不正确折叠形成的^[8],正确构象的形成需要在体外进行变性和复性。蛋白质的复性过程十分复杂,在方法上缺少一定的规律可循,特别是分子量较大以及二硫键较多的分子,复性更加困难,有的甚至根本难以复性。分子伴侣可以促进其它蛋白质的正确折叠,设想在基因工程中如果将分子伴侣基因与外源蛋白基因共存表达,可能会有效地促进外源蛋白形成正确的构象．提高其活性,减少包涵体的形成,对基因工程下游的处理带来很大方便。根 据这个思路,我们将克隆的SecB基因与重组人淋巴毒索(Lymphotoxin,简称LT)基因在同一个大肠杆菌细胞中共存表达,来研究分子伴侣SecB对外源基因表达的影响。     

大肠杆菌DsbA蛋白的氧化还原性质和构象变化

DsbA蛋白是大肠杆菌周质空间内的巯基 /二硫键氧化酶 ,主要催化底物蛋白质二硫键的形成。利用定点突变结合色氨酸类似物标记技术 ,研究了DsbA蛋白的氧化还原性质和构象变化。结果显示 :(1 )DsbA蛋白的还原态比氧化态的结构更加稳定 ,说明DsbA的强氧化性来源于氧化态构象的紧张状态 ;(2 )DsbA氧化和还原态间特殊的荧光变化主要来源于Trp76在不同状态间微观环境的差异 ;(3 )色氨酸类似物标记不会对DsbA蛋白的结构和功能产生明显的影响 ,利用1 9F NMR进一步证实了DsbA氧化还原状态间的构象变化 ,而且这种变化主要影响Trp76的局部环境 ,而对Trp1 2 6的局部环境没有太大的影响     

蛋白质二硫键异构酶家族的结构与功能

蛋白质二硫键异构酶（protein disulfide isomerase,PDI）家族是一类在内质网中起作用的巯基-二硫键氧化还原酶.它们通常含有CXXC（Cys-Xaa-Xaa-Cys,CXXC）活性位点,活性位点的两个半胱氨酸残基可催化底物二硫键的形成、异构及还原.所有PDI家族成员包含至少一个约100个氨基酸残基的硫氧还蛋白同源结构域.PDI家族的主要职能是催化内质网中新生肽链的氧化折叠,另外在内质网相关的蛋白质降解途径（ERAD）、蛋白质转运、钙稳态、抗原提呈及病毒入侵等方面也起重要作用.     

二硫键异构酶

天然二硫键的形成是许多蛋白正确折叠中的限速步骤,在稳定蛋白质构象和保持蛋白质活性方面起重要作用。讨论的二硫键异构酶是内质网中一种重要的蛋白折叠催化剂,它催化蛋白二硫键的形成和错误配对二硫键的重排,并有抑制错误折叠蛋白聚集的分子伴侣活性。PDI广泛应用于基因工程上提高外源蛋白表达水平。     

蛋白质复性研究进展

许多蛋白在大肠杆菌中高效表达时,其产物常以无活性的包含体形式存在,包含体蛋白的复性往往是制备这些蛋白的关键步骤之一,蛋白复性包括肽链折叠和分子内二硫键的氧化这两个互相影响的过程,本文综述了蛋白折叠过程的研究进展,及促进蛋白折叠和二硫键氧化的方法。     

大肠杆菌二硫键形成相关蛋白的结构、功能及其在基因工程表达外源蛋白上的应用

大肠杆菌分泌蛋白二硫键的形成是一系列蛋白协同作用的结果,主要是Dsb家族蛋白,迄今为止共发现了DsbA、DsbB、DsbC、DsbD、DsbE和DsbG。在体内,DsbA负责氧化两个巯基形成二硫键,DsbB则负责DsbA的再氧化。DsbC和DsbG负责校正DsbA导入的异常二硫键,DsbD则负责对DsbC和DsbG进行再还原,DsbE的功能与DsbD类似。除了直接和二硫键的形成相关外,DsbA、DsbC和DsbG都有分子伴侣功能。它们的分子伴侣功能独立于二硫键形成酶的活性并且对二硫键形成酶活性具有明显的促进作用。基于Dsb蛋白的功能特性,利用它们以大肠杆菌为宿主表达外源蛋白,特别是含有二硫键的蛋白,取得了很多成功的例子。本文简要介绍了这方面的进展,显示Dsb蛋白在促进外源蛋白在大肠杆菌中以可溶形式表达方面具有广阔的应用前景。     

Crystal Structure of DsbA from Corynebacterium diphtheriae and Its Functional Implications for CueP in Gram-Positive Bacteria

In Gram-negative bacteria in the periplasmic space, the dimeric thioredoxin-fold protein DsbC isomerizes and reduces incorrect disulfide bonds of unfolded proteins, while the monomeric thioredoxin-fold protein DsbA introduces disulfide bonds in folding proteins. In the Gram-negative bacteria Salmonella enterica serovar Typhimurium, the reduced form of CueP scavenges the production of hydroxyl radicals in the copper-mediated Fenton reaction, and DsbC is responsible for keeping CueP in the reduced, active form. Some DsbA proteins fulfill the functions of DsbCs, which are not present in Gram-positive bacteria. In this study, we identified a DsbA homologous protein (CdDsbA) in the Corynebacterium diphtheriae genome and determined its crystal structure in the reduced condition at 1.5 Å resolution. CdDsbA consists of a monomeric thioredoxin-like fold with an inserted helical domain and unique N-terminal extended region. We confirmed that CdDsbA has disulfide bond isomerase/reductase activity, and we present evidence that the N-terminal extended region is not required for this activity and folding of the core DsbA-like domain. Furthermore, we found that CdDsbA could reduce CueP from C. diphtheriae.     

Pathway of Oxidative Folding of a 3-Disulfide α-Lactalbumin May Resemble Either BPTI Model or Hirudin Model

Pathways of oxidative folding of disulfide proteins display a high degree of diversity and vary among two extreme models. The BPTI model is defined by limited species of folding intermediates adopting mainly native disulfide bonds. The hirudin model is characterized by highly heterogeneous folding intermediates containing mostly non-native disulfide bonds. αLA-IIIA is a 3-disulfide variant of α-lactalbumin (αLA) with a 3-D conformation essentially identical to that of intact αLA. αLA-IIIA contains 3 native disulfide bonds of αLA, two of them are located at the calcium binding β-subdomain (Cys⁶¹–Cys⁷⁷ and Cys⁷³–Cys⁹¹) and the third bridge is located within the α-helical domain of the molecule (Cys²⁸–Cys¹¹¹). We investigate here the pathway of oxidative folding of fully reduced αLA-IIIA with and without stabilization of its β-subdomain by calcium binding. In the absence of calcium, the folding pathway of αLA-IIIA was shown to resemble that of hirudin model. Upon stabilization of β-sheet domain by calcium binding, the folding pathway of αLA-IIIA exhibits a striking similarity to that of BPTI model. Three predominant folding intermediates of αLA-IIIA containing exclusively native disulfide bonds were isolated and structurally characterized. Our results further demonstrate that stabilization of subdomains in a protein may dictate its folding pathway and represent a major cause for the existing diversity in the folding pathways of the disulfide-containing proteins.     

Statistical optimization and multiple objective programming of lysozyme refolding catalyzed by recombinant DsbA in vitro

DsbA (disulfide bond formation protein A) is essential for disulfide bond formation directly affecting the nascent peptides folding to the correct conformation in vivo. In this paper, recombinant DsbA protein was employed to catalyze denatured lysozyme refolding and inhibit the aggregation of folding intermediates in vitro. Statistical methods, i.e., Plackett–Burman design and small central composite design, were adopted to screen out important factors affecting the refolding process and correlating these parameters with the refolding efficiency including both protein recovery and specific activity of refolded lysozyme. Four important parameters: initial lysozyme concentration, urea concentration, KCl concentration and GSSG (glutathione disulfide) concentration were picked out and operating conditions were optimized by introducing the effectiveness coefficient method and transforming the multiple objective programming into an ordinary constrained optimization issue. Finally, 99.7% protein recovery and 25,600 U/mg specific activity of lysozyme were achieved when 281.35 μg/mL denatured lysozyme refolding was catalyzed by an equivalent molar of DsbA at the optimal settings. The results indicated that recombinant DsbA protein could effectively catalyze the oxidized formation and reduced isomerization of intramolecular disulfide bonds in the refolding of lysozyme in vitro.     

Disulfide Linkage and Structure of Highly Stable Yeast-derived Virus-like Particles of Murine Polyomavirus

VP1 is the major coat protein of murine polyomavirus and forms virus-like particles (VLPs) in vitro. VLPs consist of 72 pentameric VP1 subunits held together by a terminal clamp structure that is further stabilized by disulfide bonds and chelation of calcium ions. Yeast-derived VLPs (yVLPs) assemble intracellularly in vivo during recombinant protein production. These in vivo assembled yVLPs differ in several properties from VLPs assembled in vitro from bacterially produced pentamers. We found several intermolecular disulfide linkages in yVLPs involving 5 of the 6 cysteines of VP1 (Cys¹¹⁵–Cys²⁰, Cys¹²–Cys²⁰, Cys¹⁶–Cys¹⁶, Cys¹²/ Cys¹⁶–Cys¹¹⁵, and Cys²⁷⁴–Cys²⁷⁴), indicating a highly coordinated disulfide network within the in vivo assembled particles involving the N-terminal region of VP1. Cryoelectron microscopy revealed structured termini not resolved in the published crystal structure of the bacterially expressed VLP that appear to clamp the pentameric subunits together. These structural features are probably the reason for the observed higher stability of in vivo assembled yVLPs compared with in vitro assembled bacterially expressed VLPs as monitored by increased thermal stability, higher resistance to trypsin cleavage, and a higher activation enthalpy of the disassembly reaction. This high stability is decreased following disassembly of yVLPs and subsequent in vitro reassembly, suggesting a role for cellular components in optimal assembly.     

Chemical synthesis of kurtoxin, a T-type calcium channel blocker

Kurtoxin isolated from the venom of scorpion, Parabuthus transvaalicus, is a 63-residue peptide with four intramolecular disulfide bonds which inhibits low-threshold T-type Ca²⁺channels. Kurtoxin was synthesized by native chemical ligation involving the coupling of (1--26)-thioester peptide and Cys²⁷-(28--63)-peptide. The former was synthesized by standard solid-phase peptide synthesis (SPPS) with Boc chemistry, while the latter was sequentially assembled from three protected segments onto a resin-bound C-terminal segment in a chloroform--phenol mixed solvent followed by deprotection reaction using HF. Each protected segment used for the coupling on a solid support was prepared on an N-[9-(hydroxymethyl)-2-fluorenyl] succinamic acid (HMFS) resin and detached from the resin by treatment with 20% Et ₃N in DMF to produce it in the form of an α-carboxylic acid. Synthetic kurtoxin obtained after the oxidative folding reaction was found to be identical with the natural product by means of several analytical procedures, and its disulfide structure was determined for the first time to be Cys¹²-Cys⁶¹, Cys¹⁶-Cys³⁷, Cys²³-Cys⁴⁴ and Cys²⁷-Cys⁴⁶ by peptide mapping, sequence analysis and mass measurements.     

A PDI-catalyzed thiol–disulfide switch regulates the production of hydrogen peroxide by human Ero1

Oxidative folding in the endoplasmic reticulum (ER) involves ER oxidoreductin 1 (Ero1)-mediated disulfide formation in protein disulfide isomerase (PDI). In this process, Ero1 consumes oxygen (O₂) and releases hydrogen peroxide (H₂O₂), but none of the published Ero1 crystal structures reveal any potential pathway for entry and exit of these reactants. We report that additional mutation of the Cys²⁰⁸–Cys²⁴¹ disulfide in hyperactive Ero1α (Ero1α-C104A/C131A) potentiates H₂O₂ production, ER oxidation, and cell toxicity. This disulfide clamps two helices that seal the flavin cofactor where O₂ is reduced to H₂O₂. Through its carboxyterminal active site, PDI unlocks this seal by forming a Cys²⁰⁸/Cys²⁴¹-dependent mixed-disulfide complex with Ero1α. The H₂O₂-detoxifying glutathione peroxidase 8 also binds to the Cys²⁰⁸/Cys²⁴¹ loop region. Supported by O₂ diffusion simulations, these data describe the first enzymatically controlled O₂ access into a flavoprotein active site, provide molecular-level understanding of Ero1α regulation and H₂O₂ production/detoxification, and establish the deleterious consequences of constitutive Ero1 activity.     

Overexpression of protein disulfide isomerase DsbA enhanced detoxification of organophosphates and enhanced detectability in the environment following degradation of pesticide residues

In many cases the expressed protein is insoluble and accumulates in so-called inclusion bodies. Several strategies have been developed to improve the solubility of the expressed protein. Disulfide isomerase DsbA could effectively assist proteins folding,both in vivo coexpressed with the target protein,and in vitro replenished as foldases.Moreover,DsbA also has the chaperone-like activity in the assistant refolding of genetically engineered inclusion bodies.Coexpression of DsbA with the target protein could lead to higher levels of soluble protein. In this work, a significantly improved, recombinant Escherichia coli has been developed to degrade the toxic organophosphorus compound, methyl parathion, to non-toxic materials by coexpression of disulfide isomerase DsbA and Methyl Parathion Hydrolase (MPH)-GFP fusion protein. Whole cells expressing DsbA had significant enhancement of MPH activity compared to DsbA-free system and could be easily detected.     

Spectroscopic, immunochemical, and thermodynamic properties of carboxymethyl(Cys6, Cys127)-hen egg white lysozyme

A three-disulfide form of hen egg white lysozyme with Cys⁶ and Cys¹²⁷ blocked by carboxymethyl groups was prepared, purified, and characterized for eventual use in protein folding experiments. Trypsin digestion followed by proline-specific endopeptidase digestion facilitated the unambiguous assignment of the disulfide bond pairings and the modified residues in this derivative. 3SS-lysozyme demonstrated nearly full enzymatic activity at itspH optimum,pH 5.5. The 3SS-lysozyme derivative and unmodified lysozyme were shown to be identical by CD spectroscopy atpH 3.6. Immunochemical binding assays demonstrated that the conformation of lysozyme was perturbed predominantly only locally by breaking and blocking the disulfide bond between Cys⁶ and Cys¹²⁷. Both 3SS-lysozyme and unmodified lysozyme exhibited reversible thermally induced transitions atpH 2.0 but theT _m of 3SS-lysozyme, 18.9°C, was found to be 34° lower than that of native lysozyme under the same conditions. The conformational chemical potential of the denatured form of unmodified lysozyme was determined from the transition curves to be approximately 6.7 kcal/mol higher than that of the denatured form of 3SS-lysozyme, atpH 2.0 and 35°C, if the conformational chemical potential for the folded forms ofboth 3SS-lysozyme and unmodified lysozyme is arbitrarily assumed to be 0.0 kcal/mol. A calculation of the increase in the theoretical loop entropy of denatured 3SS-lysozyme resulting from the cleavage of the Cys⁶-Cys¹²⁷ disulfide bond, however, yielded a value of only 5.4 kcal/mol for the difference in conformational chemical potential. This suggests that, in addition to the entropic component, there is also an enthalpic contribution to the difference in the conformational chemical potential corresponding to approximately 1.3 kcal/mol. Thus, it is concluded that the reduction and blocking of the disulfide bond between Cys⁶ and Cys¹²⁷ destabilizes 3SS-lysozyme relative to unmodified lysozyme predominantly by stabilizing the denatured conformation by increasing its chain entropy.Cornell Biotechnology Army Research Office Predoctoral Fellow, 1986–1989.     

Deciphering Structural and Functional Roles of Individual Disulfide Bonds of the Mitochondrial Sulfhydryl Oxidase Erv1p

Erv1p is a FAD-dependent sulfhydryl oxidase of the mitochondrial intermembrane space. It contains three conserved disulfide bonds arranged in two CXXC motifs and one CX₁₆C motif. Experimental evidence for the specific roles of the individual disulfide bonds is lacking. In this study, structural and functional roles of the disulfides were dissected systematically using a wide range of biochemical and biophysical methods. Three double cysteine mutants with each pair of cysteines mutated to serines were generated. All of the mutants were purified with the normal FAD binding properties as the wild type Erv1p, showing that none of the three disulfides are essential for FAD binding. Thermal denaturation and trypsin digestion studies showed that the CX₁₆C disulfide plays an important role in stabilizing the folding of Erv1p. To understand the functional role of each disulfide, small molecules and the physiological substrate protein Mia40 were used as electron donors in oxygen consumption assays. We show that both CXXC disulfides are required for Erv1 oxidase activity. The active site disulfide is well protected thus requires the shuttle disulfide for its function. Although both mutants of the CXXC motifs were individually inactive, Erv1p activity was partially recovered by mixing these two mutants together, and the recovery was rapid. Thus, we provided the first experimental evidence of electron transfer between the shuttle and active site disulfides of Erv1p, and we propose that both intersubunit and intermolecular electron transfer can occur.Disulfide bonds play very important roles in the structure and function of many proteins by stabilizing protein folding and/or acting as thiol/disulfide redox switches. The process of disulfide formation is catalyzed by dedicated enzymes in vivo (1–4). Erv1p is a FAD-dependent sulfhydryl oxidase located in the Saccharomyces cerevisiae mitochondrial intermembrane space (4–6). It is an essential component of the redox regulated Mia40/Erv1 import and assembly pathway used by many of the cysteine-containing intermembrane space proteins, such as members of the “small Tim” and Cox17 families (7–10). Upon import of a Cys-reduced substrate, Mia40 interacts with the substrate via intermolecular disulfide bond and shuttles a disulfide to its substrate. Although oxidized Mia40 promotes disulfide bond formation in the substrates, Erv1p functions in catalyzing reoxidation of the reduced Mia40 and/or release of the substrate (11–13).The common features for the FAD-dependent sulfhydryl oxidases are that the enzymes can catalyze the electron transfer from substrate molecules (e.g. protein thiols) through the noncovalent bound FAD cofactor to molecular oxygen or oxidized cytochrome c (14). The sulfhydryl oxidases can be divided into three groups: Ero1 enzymes, multidomain quiesin sulfhydryl oxidases, and single domain Erv (essential for respiration and vegetative growth)/ALR proteins. The yeast Ero1p and the mammalian homologues (Ero1α and Ero1β) are large flavoenzymes present in the ER with at least five disulfide bonds, but only two of the disulfide bonds are conserved. The conserved cysteines are essential for the catalytic activity of Ero1p forming the active site CXXC and shuttle disulfide CX₄C, respectively (15, 16). Furthermore, nonconserved disulfide bonds have been shown recently to be important in regulating the activity of both yeast and mammalian Ero1 (17–19). The second group of oxidases, the multidomain quiesin sulfhydryl oxidases, have important functions in higher eukaryotes (14, 20). Quiesin sulfhydryl oxidases consist of an Erv/ALR module fused to one or more thioredoxin-like domains with two conserved CXXC motifs in the Erv/ALR module. Quiesin sulfhydryl oxidase enzymes are found in many subcellular and extracellular locations, but not in mitochondria. Instead, single domain Erv/ARL enzymes of the third group are found in the 7mitochondria of many eukaryotic cells (21). Erv1p belongs to this single domain Erv/ARL family, which includes the human mitochondrial ARL, plant AtErv1, and yeast Erv2p of the ER lumen.The Erv/ARL enzymes are characterized by a highly conserved central catalytic core of ∼100 amino acids, which includes an active site CXXC motif (Cys¹³⁰–Cys¹³³ for Erv1p), CX₁₆C disulfide bond (Cys¹⁵⁹–Cys¹⁷⁶ for Erv1p), and residues involved in FAD binding (Fig. 1A). Based on the partial crystal structure data of Erv2p (22) and AtErv1 (23), the catalytic core of Erv proteins contains a four-helix bundle forming the noncovalent FAD-binding site with the active site CXXC in close proximity to the isoalloxazine ring of FAD. In addition, the long range CX₁₆C disulfide bond of the Erv proteins brings the short fifth helix to the four-helix bundle in proximity to the adenine ring of FAD (Fig. 1A). Thus, the CX₁₆C disulfide bond is proposed to play a structural role in stabilizing the FAD binding and/or protein folding, but direct experimental evidence to verify the roles is lacking. Apart from the catalytic core, the other parts of the proteins seem flexible and unfolded. Importantly, all members of the Erv/ALR family have at least an additional disulfide bond located in the nonconserved N- or C-terminal region to the catalytic core (Fig. 1B), which is hypothesized as a shuttle disulfide based on the partial crystal structure of Erv2 (22). The hypothesized shuttle disulfide of Erv2p CXC and AtErv1 CX₄C are located in the C terminus, but Erv1p (Cys³⁰–Cys³³) and ALR have a CXXC shuttle disulfide located N-terminal to the catalytic core. Furthermore, structural and chemical data have suggested that Erv/ARL enzymes form homodimer or oligomers in the presence or absence of intermolecular disulfide bonds (5, 23, 24).Open in a separate windowFIGURE 1.Structure and conserved Cys motifs of Erv/ALR enzymes. A, modeled structures of the conserved central catalytic core domain of Erv1p dimer based on the crystal structure data of AtErv1 (Protein Data Bank accession number 2HJ3, residues 73–173, the helix 1 starts with residue 75). The helices of the four-helix bundle and the short fifth helix are labeled from 1 to 5. The two disulfides are shown as yellow spheres, and the cofactor FAD is in red. The Cys¹³⁰–Cys¹³³ is the redox active disulfide located closely to the isoalloxazine ring of FAD. The N and C termini were labeled as N and C, respectively. The structure was generated using Pymol program. B, schematic of the primary structure of yeast, plant, and human sulfhydryl oxidase with the conserved Cys motifs. The conserved central catalytic core regions are shown as black bars, and the nonconserved regions are in gray.Yeast mitochondrial Erv1p contains a total of six Cys residues forming three pairs of disulfide bonds (residues 30–33, 130–133, and 159–176) as described above. Previous studies with single Cys mutants showed that although all three disulfide bonds are essential for Erv1p function in vivo, only Cys¹³⁰–Cys¹³³ disulfide is required for the oxidase activity of Erv1p in vitro (24). The conclusion that only Cys¹³⁰–Cys¹³³ disulfide is required for Erv1p oxidase activity in vitro was based on a study using the artificial substrate DTT² as the electron donor. Abnormal color changes were observed for some of the single Cys mutants of Erv1p in the previous study that were probably caused by protein misfolding or formation of non-native disulfides because of the presence of a redox active but unpaired Cys. It is clear that Cys¹³⁰–Cys¹³³ is the active site disulfide; however, experimental evidence for the role of Cys³⁰–Cys³³ disulfide is lacking, and the specific role played by the unique CX₁₆C motif of Erv proteins is unknown.In this study, we dissected the structural and functional roles of all three individual disulfides of Erv1p systematically. To avoid misfolding via unpaired Cys, three double Cys mutants of Erv1p were generated with each of the disulfides mutated to serines. All three mutants were successfully purified with the normal FAD binding properties of the wild type (WT) Erv1p. Various biophysical and biochemical methods were used to study the folding and oxidase activity of the WT and Erv1p mutants. Both artificial and the natural substrate (Mia40) of Erv1p were used as electron donors to understand the functional mechanism of Erv1p. Our results show that both the first (Cys³⁰–Cys³³) and second (Cys¹³⁰–Cys¹³³) disulfides are essential for Erv1 oxidase activity in vitro. Although none of the three disulfides are essential for FAD binding, the third disulfide (Cys¹⁵⁹–Cys¹⁷⁶) plays an important role in stabilizing the folding of Erv1p. More importantly, this study provided direct experimental evidence to show that Cys³⁰–Cys³³ functionally acts as a shuttle disulfide passing electrons to the active site Cys¹³⁰–Cys¹³³ disulfide. Moreover, the electron transfer seems to occur through both intersubunit and intermolecular interactions.