**4. Disulfide-bond research in the post-genomic sequencing era**

Since 2008, the cost of genome sequencing has declined faster than predicted by Moore's Law [64]. Currently, the cost of sequencing a genome is ~\$1500, and the lofty \$1000/genome goal is within reach. Due to the radical drop in DNA sequencing costs, a multitude of laboratories and private and government institutions have completed the sequencing of approximately 30,000 bacterial genomes [65]. This wealth of data is currently being used for a variety of biotechnological and clinical purposes including diagnostics, public health benefits, and biosurveillance/epidemiological studies [66, 67]. Accordingly, we have termed this time period as the "post-genomic sequencing era" to represent research that uses sequenced genomes, metagenomes, and environmental samples to search for novel enzymes and pathways and to predict the redox biology of bacteria.

### **4.1. Hunting for new disulfide-bond forming enzymes in the genomic landscape**

One of the first examples of the use of sequenced genomes to predict and identify novel disulfide-bond forming pathways was conducted by Todd Yeates and colleagues [68–70]. They hypothesized that organisms rich in disulfide-bonded proteins would have a propensity to encode for proteins with an even number of cysteine residues, since an odd number might cause formation of aberrant disulfide bonds. This conjecture was based on the observation that the predicted open reading frames (ORFs) of the hyperthermophilic *Pyrobaculum aerophilum* and *Aeropyrum pernix* species are strongly biased toward an even number of cysteines [70]. Since then, they have expanded their analysis to show that hyperthermophilic members of the Crenarchaeota branch all contain a multitude of disulfide-bonded proteins [68]. Mass spectrometric analysis of the proteome of *Sulfolobus solfataricus* revealed the majority of cysteines to be disulfide bonded [71], and several disulfide-bonded proteins were identified using 2D gel analysis of lysates of *P. aerophilum* [72]. The presence of a high number of disulfide bond-containing proteins in hyperthermophilic Crenarchaeota suggested these bacteria possess an undiscovered method of disulfide-bond maintenance. Indeed, experimental evidence of such a system was obtained from the *in vitro* characterization of protein disulfide oxidoreductases (PDO) from *Pyrococcus furiosus* [73], *Aquifex aeolicus* [74], *A. pernix* [75], and *S. solfataricus* [76]. PDOs have been shown to be functional homologs of PDI and DsbC, that exhibit reduction, oxidation, and isomerization of disulfide bonds. Although there is growing evidence that the cytoplasm of Crenarchaeota is more amenable to disulfide-bond formation, the exact mechanism and the enzymes involved remain to be elucidated *in vivo*.

(PspE) with a single cysteine that can promote disulfide-bond formation in a strain completely

From Biology to Biotechnology: Disulfide Bond Formation in *Escherichia coli*

http://dx.doi.org/10.5772/67393

367

Heavy metals such as copper or cadmium can oxidize thiol groups in periplasmic proteins, resulting in misfolding of proteins containing cysteines and, in some cases, leading to death [83]. DsbC can reduce and refold proteins that were misoxidized by such metals and is therefore necessary to protect cells from copper and cadmium-induced oxidative damage. This phenotype was used to select for strains containing mutant DsbG proteins that have gained the ability to isomerize misoxidized proteins [84]. In another heavy metal screen, cells lacking the *dsbA* gene were screened for cadmium resistance to select for mutant DsbB that can bypass the need for DsbA [85]. The mutant DsbB proteins were able to oxidize DsbC and thus

A blue/white screen was developed using a mutant alkaline phosphatase (*phoA\**) that required DsbC for its correct folding and activity. Unlike DsbC, DsbG cannot isomerize misoxidized PhoA\*. Mutants of *dsbG* were selected for their gained ability to isomerize PhoA\*, resulting in the first *in vivo* screen that directly detected disulfide-bond isomerization of a single protein. This screen permitted the identification of key residues that converted a sulfenic acid reductase (DsbG) into a disulfide-bond isomerase whose activity increased the cells' resistance to copper. Searching the genomes of sequenced prokaryotes, homologs of DsbG were discovered to naturally have the key residues identified through the *phoA*\* screen. Interestingly, these naturally existing homologs were also capable of protecting cells against copper toxicity. Thus, through the identification of these key residues, activities of homologs can be pre-

The study of disulfide-bond formation has grown and matured significantly since the discovery of DsbA in 1991 [28]. Subsequently, the Dsb pathway in the model organism *E. coli* has been studied in great detail both *in vivo* and *in vitro,* and many novel and interesting mutants and suppressors have been identified using various *in vivo* screens. These new enzymes should have applications

Both the pharmaceutical and the biotechnological industries are extremely interested in disulfide-bonded proteins. Most eukaryotic cell surface and secreted proteins are rich in disulfide bonds due to the increased stability they confer, making these proteins attractive candidates as therapeutics (also known as biologics). For example, the first recombinant biologic was the hormone insulin, which was introduced by Eli Lilly in 1982, and the most profitable biologic is the antibody Humira (adalimumab), both of which are disulfide-bonded proteins [87]. Between 1982 and 2013, approximately 100 recombinant protein therapeutics have been approved by the FDA, of which more than one-third are disulfide-bonded proteins (in par-

Currently, antibodies represent the fastest growing category of biologics. Their specificity to therapeutic targets, ability to induce or inhibit immune response, and favorable pharmacokinetic profiles within the human body make them attractive therapeutics. The first therapeutic

in both biotechnology and the pharmaceutical industry as detailed in the next section.

**4.3. Biotechnological applications of disulfide-bonded proteins**

lacking the *dsb* pathway [82].

promote disulfide-bond formation.

dicted and tested [86].

ticular monoclonal antibodies) [88].

The method of predicting redox biology of organisms by simply analyzing the cysteine content of the predicted ORFs from sequenced genomes was expanded to all prokaryotic organisms with known genome sequences. By separating the predicted proteome into two subgroups—proteins predicted to be exported and those that remained in the cytoplasm this bioinformatic method was further developed to predict whether the periplasmic space was oxidizing or reducing [77]. This method led to the observation that some bacteria predicted to have an oxidizing periplasm encode a homolog of DsbA but lack a homolog of its partner DsbB. A closer look at these strains revealed that the DsbA homolog in *Mycobacterium* was a fusion protein to vitamin K epoxide reductase (VKOR) [77]. Characterization of bacterial VKOR homologs confirmed that VKOR can indeed functionally replace DsbB in certain organisms [78, 79]. To our knowledge, this was the first use of genomic data to mine for new oxidoreductases, leading to the discovery of VKOR as a functional homolog of DsbB.

### **4.2. Selecting for new oxidoreductases using living bacteria**

The advent of modern biomolecular tools, in conjunction with classical bacterial genetic screens, has led to the discovery of novel enzymes, yielded many new insights into biochemical pathways, and elucidated molecular mechanisms. The discovery that disulfide bonds are not formed spontaneously but are, in fact, formed catalytically by the enzyme DsbA was a serendipitous discovery using a blue/white screen for secretion defects [28]. The malF-lacZ fusion has been used to not only discover DsbA [28] but also mutants of DsbA with various kinetic properties [31]. Since then, many other genetic screens have been developed to specifically detect the activity of an oxidoreductase in *E. coli*. These screens, described briefly below, allow for the selection of gene products whose activities permit the growth of strains in the absence of a *dsb* component. Characterization of mutant strains revealed insight into the molecular machinery of disulfide-bond formation and highlighted the plasticity of the dsb machinery. A few key mutations could convert a dedicated reductase into an oxidase or create novel pathways to maintain cell viability.

FlgI is a protein component of the flagellar machinery and requires a disulfide bond for its correct folding and activity [80]. Strains that have a functional disulfide-bond forming pathway are motile, while those with defects in disulfide-bond formation are not. By simply spotting bacteria incapable of forming disulfide bonds on dilute agar, researchers are able to screen and select for bacteria that have gained the ability to form disulfide bonds, since they become motile and swim away from the center. This phenotype has been used to characterize and select for new disulfide bond oxidases, such as selecting for mutant thioredoxins possessing a new mechanism of disulfide-bond formation in the periplasm [81]. In another approach, researchers screened a multicopy plasmid library of *E. coli* and selected a rhodanese protein (PspE) with a single cysteine that can promote disulfide-bond formation in a strain completely lacking the *dsb* pathway [82].

evidence of such a system was obtained from the *in vitro* characterization of protein disulfide oxidoreductases (PDO) from *Pyrococcus furiosus* [73], *Aquifex aeolicus* [74], *A. pernix* [75], and *S. solfataricus* [76]. PDOs have been shown to be functional homologs of PDI and DsbC, that exhibit reduction, oxidation, and isomerization of disulfide bonds. Although there is growing evidence that the cytoplasm of Crenarchaeota is more amenable to disulfide-bond formation,

The method of predicting redox biology of organisms by simply analyzing the cysteine content of the predicted ORFs from sequenced genomes was expanded to all prokaryotic organisms with known genome sequences. By separating the predicted proteome into two subgroups—proteins predicted to be exported and those that remained in the cytoplasm this bioinformatic method was further developed to predict whether the periplasmic space was oxidizing or reducing [77]. This method led to the observation that some bacteria predicted to have an oxidizing periplasm encode a homolog of DsbA but lack a homolog of its partner DsbB. A closer look at these strains revealed that the DsbA homolog in *Mycobacterium* was a fusion protein to vitamin K epoxide reductase (VKOR) [77]. Characterization of bacterial VKOR homologs confirmed that VKOR can indeed functionally replace DsbB in certain organisms [78, 79]. To our knowledge, this was the first use of genomic data to mine for new

the exact mechanism and the enzymes involved remain to be elucidated *in vivo*.

366 *Escherichia coli* Escherichia coli - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications

oxidoreductases, leading to the discovery of VKOR as a functional homolog of DsbB.

The advent of modern biomolecular tools, in conjunction with classical bacterial genetic screens, has led to the discovery of novel enzymes, yielded many new insights into biochemical pathways, and elucidated molecular mechanisms. The discovery that disulfide bonds are not formed spontaneously but are, in fact, formed catalytically by the enzyme DsbA was a serendipitous discovery using a blue/white screen for secretion defects [28]. The malF-lacZ fusion has been used to not only discover DsbA [28] but also mutants of DsbA with various kinetic properties [31]. Since then, many other genetic screens have been developed to specifically detect the activity of an oxidoreductase in *E. coli*. These screens, described briefly below, allow for the selection of gene products whose activities permit the growth of strains in the absence of a *dsb* component. Characterization of mutant strains revealed insight into the molecular machinery of disulfide-bond formation and highlighted the plasticity of the dsb machinery. A few key mutations could convert a dedicated reductase into an oxidase or create

FlgI is a protein component of the flagellar machinery and requires a disulfide bond for its correct folding and activity [80]. Strains that have a functional disulfide-bond forming pathway are motile, while those with defects in disulfide-bond formation are not. By simply spotting bacteria incapable of forming disulfide bonds on dilute agar, researchers are able to screen and select for bacteria that have gained the ability to form disulfide bonds, since they become motile and swim away from the center. This phenotype has been used to characterize and select for new disulfide bond oxidases, such as selecting for mutant thioredoxins possessing a new mechanism of disulfide-bond formation in the periplasm [81]. In another approach, researchers screened a multicopy plasmid library of *E. coli* and selected a rhodanese protein

**4.2. Selecting for new oxidoreductases using living bacteria**

novel pathways to maintain cell viability.

Heavy metals such as copper or cadmium can oxidize thiol groups in periplasmic proteins, resulting in misfolding of proteins containing cysteines and, in some cases, leading to death [83]. DsbC can reduce and refold proteins that were misoxidized by such metals and is therefore necessary to protect cells from copper and cadmium-induced oxidative damage. This phenotype was used to select for strains containing mutant DsbG proteins that have gained the ability to isomerize misoxidized proteins [84]. In another heavy metal screen, cells lacking the *dsbA* gene were screened for cadmium resistance to select for mutant DsbB that can bypass the need for DsbA [85]. The mutant DsbB proteins were able to oxidize DsbC and thus promote disulfide-bond formation.

A blue/white screen was developed using a mutant alkaline phosphatase (*phoA\**) that required DsbC for its correct folding and activity. Unlike DsbC, DsbG cannot isomerize misoxidized PhoA\*. Mutants of *dsbG* were selected for their gained ability to isomerize PhoA\*, resulting in the first *in vivo* screen that directly detected disulfide-bond isomerization of a single protein. This screen permitted the identification of key residues that converted a sulfenic acid reductase (DsbG) into a disulfide-bond isomerase whose activity increased the cells' resistance to copper. Searching the genomes of sequenced prokaryotes, homologs of DsbG were discovered to naturally have the key residues identified through the *phoA*\* screen. Interestingly, these naturally existing homologs were also capable of protecting cells against copper toxicity. Thus, through the identification of these key residues, activities of homologs can be predicted and tested [86].

The study of disulfide-bond formation has grown and matured significantly since the discovery of DsbA in 1991 [28]. Subsequently, the Dsb pathway in the model organism *E. coli* has been studied in great detail both *in vivo* and *in vitro,* and many novel and interesting mutants and suppressors have been identified using various *in vivo* screens. These new enzymes should have applications in both biotechnology and the pharmaceutical industry as detailed in the next section.

### **4.3. Biotechnological applications of disulfide-bonded proteins**

Both the pharmaceutical and the biotechnological industries are extremely interested in disulfide-bonded proteins. Most eukaryotic cell surface and secreted proteins are rich in disulfide bonds due to the increased stability they confer, making these proteins attractive candidates as therapeutics (also known as biologics). For example, the first recombinant biologic was the hormone insulin, which was introduced by Eli Lilly in 1982, and the most profitable biologic is the antibody Humira (adalimumab), both of which are disulfide-bonded proteins [87]. Between 1982 and 2013, approximately 100 recombinant protein therapeutics have been approved by the FDA, of which more than one-third are disulfide-bonded proteins (in particular monoclonal antibodies) [88].

Currently, antibodies represent the fastest growing category of biologics. Their specificity to therapeutic targets, ability to induce or inhibit immune response, and favorable pharmacokinetic profiles within the human body make them attractive therapeutics. The first therapeutic monoclonal antibody product, Orthoclone OKT3 (muromonab-CD3), was FDA approved in 1986. Since then, research and development of biologics has led to many successful therapeutics, with projected sales expected to reach nearly \$125 billion by 2020 [89] (see **Table 1** for top 11 best-selling biologics in 2013 [90]). The production of antibodies for therapeutic applications is a well-established pipeline dominated by the use of Chinese hamster ovary (CHO) cells or hybridomas. However, identifying, characterizing, and engineering therapeutic antibodies are still expensive, time-consuming, and effortful endeavors, leaving room for these aspects of biologic development to be streamlined.

The use of *E. coli* as the most popular host for recombinant engineering of proteins stems from the bevy of powerful genetic tools available, its cost effectiveness, and the short time frames required for both its growth and genetic experiments. The periplasm, where conditions favor oxidized proteins, was the clear compartment to express various antibody fragments, including full-length antibodies, versus the reducing conditions of the cytoplasm [91, 92]. The periplasmic space remains an attractive alternative for the production of disulfidebonded proteins whose presence/activities may be toxic when expressed in the cytoplasm [93]. However, translocation of the target protein across the inner membrane to the periplasm can be problematic and may require extensive optimization of both the expression conditions and the targeting signal sequence. Furthermore, the lack of ATP in the periplasm makes it an energy-poor environment for proteins that require ATP-dependent chaperones for their folding. The cytoplasm is therefore a more suitable compartment for high-yielding protein production. It also obviates the problem of crossing the membrane and is rich in ATP, chap-

From Biology to Biotechnology: Disulfide Bond Formation in *Escherichia coli*

http://dx.doi.org/10.5772/67393

369

With the introduction of the Δ*trxB*, *gor* engineered strains of *E. coli* [94, 95], it is now possible to not only express various antibody fragments but also full-length antibodies in the cytoplasm [96]. Yet, the lack of N-linked glycosylation in *E. coli* has hampered its use in the production of therapeutic immunoglobulins (IgGs), although a few examples of *E. coli-*produced therapeutic antibody fragments can be found, such as the Fab' fragment named Lucentis (ranibizumab) against age-related macular degeneration [97]. The discovery of mutations in the Fc portion of IgG that circumvent the dependency on glycosylation for effective interaction with its cognate Fcγ receptor [98] opened the path to potential therapeutic applications of *E. coli-*expressed IgG [96]. Though *E. coli* is currently not as established as CHO or hybridoma cell lines for the production of therapeutic IgG, it is slowly becoming a more common host for the production of antibodies. Other *E. coli-*based technologies, such as phage display, have had extensive use in the discovery and engineering of antibodies, both for the biotech and the pharmaceutical industries. The use of phage display technology to identify novel antibodies of therapeutic targets, such as the HIV virus coat protein, was first described in 1991 [99]. Since then, phage display has been used to develop novel antibody-based applications. For example, the antibody Humira went through extensive engineering using this technique to create an effective biologic [100].

One key feature of disulfide bonds is their ability to increase the thermostability of proteins by decreasing the number of conformations a protein can attain and thus lowering the conformational entropy of a protein. Secreted proteins leave the protective environment of the cell cytoplasm, and they are rich in disulfide bonds which help to increase their extracellular half-lives. These enzymes are of significant utility in the biotech industry where high-temperature processes are often used. In some cases, disulfide bonds have been introduced into such enzymes to increase their thermostability [101]. Early investigations into the effects of engineered disulfide bonds were performed on phage lambda repressor [102], T4 lysozyme [103], and subtilisin [104], and later were expanded to antibodies [105] and other proteins used in the biotechnology industry. For example, the disulfide bond engineered into the extracellular ribonuclease (barnase) from *Bacillus amyloliquefaciens* unfolds 20 times slower than wild type and 170 times

erones, and folding factors.

**4.4. Engineering disulfide bonds**


Abbreviations: mAb, monoclonal antibody; RA, rheumatoid arthritis; PA, psoriatic arthritis; UC, ulcerative colitis; AA, anaplastic astrocytoma; GBM, glioblastoma multiforme. Adapted from Ref. [90].

**Table 1.** The top 10 best selling biologics in 2013. Of these 11 biologics, five are antibody-based therapeutics, indicated by the mAb under molecule type.

The use of *E. coli* as the most popular host for recombinant engineering of proteins stems from the bevy of powerful genetic tools available, its cost effectiveness, and the short time frames required for both its growth and genetic experiments. The periplasm, where conditions favor oxidized proteins, was the clear compartment to express various antibody fragments, including full-length antibodies, versus the reducing conditions of the cytoplasm [91, 92]. The periplasmic space remains an attractive alternative for the production of disulfidebonded proteins whose presence/activities may be toxic when expressed in the cytoplasm [93]. However, translocation of the target protein across the inner membrane to the periplasm can be problematic and may require extensive optimization of both the expression conditions and the targeting signal sequence. Furthermore, the lack of ATP in the periplasm makes it an energy-poor environment for proteins that require ATP-dependent chaperones for their folding. The cytoplasm is therefore a more suitable compartment for high-yielding protein production. It also obviates the problem of crossing the membrane and is rich in ATP, chaperones, and folding factors.

With the introduction of the Δ*trxB*, *gor* engineered strains of *E. coli* [94, 95], it is now possible to not only express various antibody fragments but also full-length antibodies in the cytoplasm [96]. Yet, the lack of N-linked glycosylation in *E. coli* has hampered its use in the production of therapeutic immunoglobulins (IgGs), although a few examples of *E. coli-*produced therapeutic antibody fragments can be found, such as the Fab' fragment named Lucentis (ranibizumab) against age-related macular degeneration [97]. The discovery of mutations in the Fc portion of IgG that circumvent the dependency on glycosylation for effective interaction with its cognate Fcγ receptor [98] opened the path to potential therapeutic applications of *E. coli-*expressed IgG [96]. Though *E. coli* is currently not as established as CHO or hybridoma cell lines for the production of therapeutic IgG, it is slowly becoming a more common host for the production of antibodies. Other *E. coli-*based technologies, such as phage display, have had extensive use in the discovery and engineering of antibodies, both for the biotech and the pharmaceutical industries. The use of phage display technology to identify novel antibodies of therapeutic targets, such as the HIV virus coat protein, was first described in 1991 [99]. Since then, phage display has been used to develop novel antibody-based applications. For example, the antibody Humira went through extensive engineering using this technique to create an effective biologic [100].

### **4.4. Engineering disulfide bonds**

monoclonal antibody product, Orthoclone OKT3 (muromonab-CD3), was FDA approved in 1986. Since then, research and development of biologics has led to many successful therapeutics, with projected sales expected to reach nearly \$125 billion by 2020 [89] (see **Table 1** for top 11 best-selling biologics in 2013 [90]). The production of antibodies for therapeutic applications is a well-established pipeline dominated by the use of Chinese hamster ovary (CHO) cells or hybridomas. However, identifying, characterizing, and engineering therapeutic antibodies are still expensive, time-consuming, and effortful endeavors, leaving room for these

**Approved indication(s) 2013 worldwide** 

psoriasis, ankylosing spondylitis, UC

cell lymphocytic lymphoma, non-Hodgkin's lymphoma, antineutrophil cytoplasmic antibodies-associated vasculitis, indolent non-Hodgkin's lymphoma, diffuse large

mAb RA, Crohn's disease, psoriasis, UC, ankylosing

renal cell cancer, brain cancer (malignant

Peptide Multiple sclerosis 4356

Multiple myeloma, myelodysplastic syndrome,

**sales (\$ millions)**

10,659

8739

7593

7500

6962

6747

4693

4281

aspects of biologic development to be streamlined.

**Molecule type**

368 *Escherichia coli* Escherichia coli - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications

Humira *(adalimumab)* AbbVie mAb RA, juvenile RA, Crohn's disease, PA,

Enbrel *(etanercept)* Amgen Protein RA, psoriasis, ankylosing spondylitis, PA

Rituxan (*rituximab*) Roche mAb RA, chronic, lymphocytic leukemia/small

Avastin (*bevacizumab*) Roche mAb Colorectal cancer, non-small cell lung cancer,

molecule

molecule

anaplastic astrocytoma; GBM, glioblastoma multiforme. Adapted from Ref. [90].

juvenile RA

Sanofi Peptide Diabetes mellitus type I, diabetes mellitus type II

B-cell lymphoma

spondylitis, PA

glioma; AA and GBM)

Roche mAb Breast cancer, gastric cancer 6558

Chronic myelogenous leukemia, gastrointestinal stromal tumor, acute lymphocytic leukemia, hypereosinophilic syndrome, mastocytosis, dermatofibrosarcoma protuberans, myelodysplastic syndrome,

myeloproliferative disorders

Amgen Protein Neutropenia/leukopenia 4392

mantle cell lymphoma

Abbreviations: mAb, monoclonal antibody; RA, rheumatoid arthritis; PA, psoriatic arthritis; UC, ulcerative colitis; AA,

**Table 1.** The top 10 best selling biologics in 2013. Of these 11 biologics, five are antibody-based therapeutics, indicated

**company**

**Name Lead** 

Remicade (*infliximab*) Johnson &

Johnson

Teva Pharmaceutical

Celgene Small

Gleevec (*imatinib*) Novartis Small

Lantus (*insulin glargine*)

Herceptin (*trastuzumab*)

Neulasta (*pegfilgrastim*)

*acetate*)

Revlimid (*lenalidomide*)

Copaxone (*glatiramer* 

by the mAb under molecule type.

One key feature of disulfide bonds is their ability to increase the thermostability of proteins by decreasing the number of conformations a protein can attain and thus lowering the conformational entropy of a protein. Secreted proteins leave the protective environment of the cell cytoplasm, and they are rich in disulfide bonds which help to increase their extracellular half-lives. These enzymes are of significant utility in the biotech industry where high-temperature processes are often used. In some cases, disulfide bonds have been introduced into such enzymes to increase their thermostability [101]. Early investigations into the effects of engineered disulfide bonds were performed on phage lambda repressor [102], T4 lysozyme [103], and subtilisin [104], and later were expanded to antibodies [105] and other proteins used in the biotechnology industry. For example, the disulfide bond engineered into the extracellular ribonuclease (barnase) from *Bacillus amyloliquefaciens* unfolds 20 times slower than wild type and 170 times slower than the reduced protein [106]. It is also possible to engineer an interchain disulfide bond within two subunits to bring together the activities of two distinct enzymes [107].

involves the activity of the enzyme vitamin K epoxide reductase (VKOR), which is inhibited by the anticoagulant drug warfarin (Coumadin). Interestingly, *Mycobacterium tuberculosis (Mtb)* and other bacteria do not encode for a DsbB protein, but instead encode a homolog of VKOR. Although DsbB and VKOR exhibit little sequence similarity, they appear to be functionally similar, since VKOR can replace DsbB in both *E. coli* and cyanobacterial *ΔdsbB* strains [77, 123]. Warfarin was shown to both inhibit *Mtb*VKOR activity and bacterial growth. Furthermore, mutations in the VKOR protein from warfarin-resistant *Mtb* mutants were mapped to nearly identical locations in mutant VKORs from patients who require higher effective doses of warfarin, indicating the drug likely inhibits bacterial and human VKORs in similar manners [34]. These findings, in conjunction with the severe growth defects observed in *Mtb*VKOR homolog deletion strains, suggested that stronger inhibitors of *Mtb*VKOR could

Promising small molecule inhibitors of bacterial Dsb proteins have been identified using fragment-based lead discovery (FBLD) [124]. FBLD identifies small molecule fragments that weakly bind to a target of interest. Through many rounds of iterative combinations of such fragments and high-throughput screening, candidate molecules with higher binding affinities for the target are created, leading to possible drug candidates. Using a detergent-solubilized *Ec*DsbB

to and inhibition of *Ec*DsbB, yielding eight fragments exhibiting IC50 values of 7–170 μM. The eight fragments were divided into two groups based on their molecular scaffolds and hypothesized mechanisms of inhibition: blocking of quinone binding and blocking of both quinone and *Ec*DsbA binding to DsbB [124]. A further study improved the IC50 value of a candidate molecule to 1.1 μM through additional rounds of FBLD. This molecule inhibited both *Ec*DsbA and DsbB through covalent modification of active site cysteine residue in each protein with a propionyl group, thereby abrogating their ability to form disulfide bonds. The molecule also exhibited a degree of selectivity for DsbA and DsbB proteins, since it was shown to have no

Through the use of high-throughput blue/white screening, six additional small molecule inhibitors of *Ec*DsbB were identified from a pool of approximately 52,000 compounds. These six molecules all contained a pyridazinone ring and exhibited a degree of selectivity for *Ec*DsbB, since they were unable to inhibit the *Mtb*VKOR homolog described above. Interestingly, the molecules inhibited DsbB enzymes from other Gram-negative pathogens, including *V. cholerae, Haemophilus influenzae, Salmonella typhimurium, Klebsiella pneumoniae, Francisella tularensis, Acinetobacter baumannii*, and *P. aeruginosa*, to varying degrees [126].

In addition to small molecules, larger peptides capable of inhibiting the formation of the DsbA-DsbB complex have been developed. Using the crystal structure of the DsbA-DsbB complex [127], a peptide of seven amino acids corresponding to a loop of DsbB involved in docking with DsbA was identified and found to bind to *Ec*DsbA with low micromolar affinity (Kd = 13.1 ± 0.4 μM). Further engineering of this peptide resulted in a new peptide with greater affinity (Kd = 5.7 ± 0.4 μM) that also exhibited fairly potent inhibition of *Ec*DsbA oxidase activity (IC50 = 8.8 ± 1.1 μM) [128]. The studies described herein clearly show that the DsbA-DsbB protein system is an attractive and tractable target for novel antibiotic

H NMR, 1071 fragments were tested for both binding

From Biology to Biotechnology: Disulfide Bond Formation in *Escherichia coli*

http://dx.doi.org/10.5772/67393

371

be used as effective antituberculosis agents [34].

immobilized onto sepharose resin and 1

effect on human thioredoxin activity [125].

In addition to engineering disulfide bonds into proteins, the reactivity of disulfide-bond forming proteins can also be altered to provide new functionalities. For example, chimeras were created by fusing the disulfide-bond oxidase DsbA to the dimerization domain and α-helical linker derived from the bacterial proline *cis/trans* isomerase FkpA. These chimeras were capable of catalyzing the *in vivo* isomerization of misoxidized disulfide bonds with similar efficiency as that of DsbC [108]. The DsbA-FkpA chimeras also conferred modest resistance to CuCl2 , which is dependent on disulfide-bond isomerization. This resistance allowed for the selection of DsbA-FkpA mutants which were found to contain a single amino acid variation in the active site of DsbA from CPHC to CPYC. Substitution of histidine with tyrosine made the active site more DsbC-like (CGYC), which could partially explain the gain of DsbC-like isomerization activity. Interestingly, DsbC is not a substrate for the DsbA-DsbB oxidation system and does not exhibit disulfide-bond oxidase activity. However, the DsbA-FkpA chimeras exhibited both oxidase and isomerization functionalities, and *dsbA* deletion strains were partially complemented by the presence of the DsbB-dependent DsbA-FkpA chimeras [108].

### **4.5. Dsb enzymes as novel antimicrobial targets**

Many pathogenic bacteria, including *Vibrio cholerae, Pseudomonas aeruginosa, Salmonella enterica, Helicobacter pylori, Bordetella pertussis,* and *E. coli,* among others, make use of periplasmic disulfide-bonded proteins that act as virulence factors or function in processes related to their pathogenicity [109–114]. These virulence factors and pathogenic functions rely on the Dsb proteins, in particular DsbA, for proper folding. As a consequence, disruptions in the redox and isomerization activities of the dsb system partially or fully attenuate the pathogenicity of these bacterial species [115]. Specifically, maturation of toxins of *V. cholerae, B. pertussis,* and *E. coli* requires the formation of DsbA-dependent disulfide bonds [109, 113, 116, 117]. Strains of these bacteria lacking *dsbA* synthesize misfolded, misassembled, and/or unstable toxin proteins that are severely impaired or nonfunctional. Along these lines, Δ*dsbA* strains of *S. enterica* and *E. coli* lack the flagellin (FliC) protein, which is a primary constituent of the filaments of their flagella. FliC does not contain any disulfide bonds. However, due to the hierarchical assembly of the flagellum machinery, which requires several proteins with disulfide bonds to precede FliC in its biogenesis, it is thought that FliC simply is not translated or that it cannot be assembled into the organelle due to the missing disulfide bonds and/or disulfidebonded proteins [50, 118]. As a result, these Δ*dsbA* strains of *S. enterica* and *E. coli* bacteria are nonmotile and their pathogenicity is severely attenuated. Additionally, the loss of disulfide bonds in Δ*dsbA* strains of *V. cholerae* and *E. coli* affects their ability to adhere to eukaryotic cells and/or form biofilms due to defects in their pili, thereby limiting their infectivity [109, 119, 120]. All together, these studies showed that the Dsb enzymes, especially DsbA, play crucial roles in the pathogenicity of several species of bacteria, making these enzymes logical targets for novel antibiotic development.

Indeed, some research has focused on the development of small molecule inhibitors of Dsb enzymes and their homologs (reviewed in Refs. [121, 122]). In humans, blood coagulation involves the activity of the enzyme vitamin K epoxide reductase (VKOR), which is inhibited by the anticoagulant drug warfarin (Coumadin). Interestingly, *Mycobacterium tuberculosis (Mtb)* and other bacteria do not encode for a DsbB protein, but instead encode a homolog of VKOR. Although DsbB and VKOR exhibit little sequence similarity, they appear to be functionally similar, since VKOR can replace DsbB in both *E. coli* and cyanobacterial *ΔdsbB* strains [77, 123]. Warfarin was shown to both inhibit *Mtb*VKOR activity and bacterial growth. Furthermore, mutations in the VKOR protein from warfarin-resistant *Mtb* mutants were mapped to nearly identical locations in mutant VKORs from patients who require higher effective doses of warfarin, indicating the drug likely inhibits bacterial and human VKORs in similar manners [34]. These findings, in conjunction with the severe growth defects observed in *Mtb*VKOR homolog deletion strains, suggested that stronger inhibitors of *Mtb*VKOR could be used as effective antituberculosis agents [34].

slower than the reduced protein [106]. It is also possible to engineer an interchain disulfide bond within two subunits to bring together the activities of two distinct enzymes [107].

370 *Escherichia coli* Escherichia coli - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications

In addition to engineering disulfide bonds into proteins, the reactivity of disulfide-bond forming proteins can also be altered to provide new functionalities. For example, chimeras were created by fusing the disulfide-bond oxidase DsbA to the dimerization domain and α-helical linker derived from the bacterial proline *cis/trans* isomerase FkpA. These chimeras were capable of catalyzing the *in vivo* isomerization of misoxidized disulfide bonds with similar efficiency as that of DsbC [108]. The DsbA-FkpA chimeras also conferred modest resistance

selection of DsbA-FkpA mutants which were found to contain a single amino acid variation in the active site of DsbA from CPHC to CPYC. Substitution of histidine with tyrosine made the active site more DsbC-like (CGYC), which could partially explain the gain of DsbC-like isomerization activity. Interestingly, DsbC is not a substrate for the DsbA-DsbB oxidation system and does not exhibit disulfide-bond oxidase activity. However, the DsbA-FkpA chimeras exhibited both oxidase and isomerization functionalities, and *dsbA* deletion strains were partially complemented by the presence of the DsbB-dependent DsbA-FkpA chimeras [108].

Many pathogenic bacteria, including *Vibrio cholerae, Pseudomonas aeruginosa, Salmonella enterica, Helicobacter pylori, Bordetella pertussis,* and *E. coli,* among others, make use of periplasmic disulfide-bonded proteins that act as virulence factors or function in processes related to their pathogenicity [109–114]. These virulence factors and pathogenic functions rely on the Dsb proteins, in particular DsbA, for proper folding. As a consequence, disruptions in the redox and isomerization activities of the dsb system partially or fully attenuate the pathogenicity of these bacterial species [115]. Specifically, maturation of toxins of *V. cholerae, B. pertussis,* and *E. coli* requires the formation of DsbA-dependent disulfide bonds [109, 113, 116, 117]. Strains of these bacteria lacking *dsbA* synthesize misfolded, misassembled, and/or unstable toxin proteins that are severely impaired or nonfunctional. Along these lines, Δ*dsbA* strains of *S. enterica* and *E. coli* lack the flagellin (FliC) protein, which is a primary constituent of the filaments of their flagella. FliC does not contain any disulfide bonds. However, due to the hierarchical assembly of the flagellum machinery, which requires several proteins with disulfide bonds to precede FliC in its biogenesis, it is thought that FliC simply is not translated or that it cannot be assembled into the organelle due to the missing disulfide bonds and/or disulfidebonded proteins [50, 118]. As a result, these Δ*dsbA* strains of *S. enterica* and *E. coli* bacteria are nonmotile and their pathogenicity is severely attenuated. Additionally, the loss of disulfide bonds in Δ*dsbA* strains of *V. cholerae* and *E. coli* affects their ability to adhere to eukaryotic cells and/or form biofilms due to defects in their pili, thereby limiting their infectivity [109, 119, 120]. All together, these studies showed that the Dsb enzymes, especially DsbA, play crucial roles in the pathogenicity of several species of bacteria, making these enzymes logical targets

Indeed, some research has focused on the development of small molecule inhibitors of Dsb enzymes and their homologs (reviewed in Refs. [121, 122]). In humans, blood coagulation

, which is dependent on disulfide-bond isomerization. This resistance allowed for the

to CuCl2

**4.5. Dsb enzymes as novel antimicrobial targets**

for novel antibiotic development.

Promising small molecule inhibitors of bacterial Dsb proteins have been identified using fragment-based lead discovery (FBLD) [124]. FBLD identifies small molecule fragments that weakly bind to a target of interest. Through many rounds of iterative combinations of such fragments and high-throughput screening, candidate molecules with higher binding affinities for the target are created, leading to possible drug candidates. Using a detergent-solubilized *Ec*DsbB immobilized onto sepharose resin and 1 H NMR, 1071 fragments were tested for both binding to and inhibition of *Ec*DsbB, yielding eight fragments exhibiting IC50 values of 7–170 μM. The eight fragments were divided into two groups based on their molecular scaffolds and hypothesized mechanisms of inhibition: blocking of quinone binding and blocking of both quinone and *Ec*DsbA binding to DsbB [124]. A further study improved the IC50 value of a candidate molecule to 1.1 μM through additional rounds of FBLD. This molecule inhibited both *Ec*DsbA and DsbB through covalent modification of active site cysteine residue in each protein with a propionyl group, thereby abrogating their ability to form disulfide bonds. The molecule also exhibited a degree of selectivity for DsbA and DsbB proteins, since it was shown to have no effect on human thioredoxin activity [125].

Through the use of high-throughput blue/white screening, six additional small molecule inhibitors of *Ec*DsbB were identified from a pool of approximately 52,000 compounds. These six molecules all contained a pyridazinone ring and exhibited a degree of selectivity for *Ec*DsbB, since they were unable to inhibit the *Mtb*VKOR homolog described above. Interestingly, the molecules inhibited DsbB enzymes from other Gram-negative pathogens, including *V. cholerae, Haemophilus influenzae, Salmonella typhimurium, Klebsiella pneumoniae, Francisella tularensis, Acinetobacter baumannii*, and *P. aeruginosa*, to varying degrees [126].

In addition to small molecules, larger peptides capable of inhibiting the formation of the DsbA-DsbB complex have been developed. Using the crystal structure of the DsbA-DsbB complex [127], a peptide of seven amino acids corresponding to a loop of DsbB involved in docking with DsbA was identified and found to bind to *Ec*DsbA with low micromolar affinity (Kd = 13.1 ± 0.4 μM). Further engineering of this peptide resulted in a new peptide with greater affinity (Kd = 5.7 ± 0.4 μM) that also exhibited fairly potent inhibition of *Ec*DsbA oxidase activity (IC50 = 8.8 ± 1.1 μM) [128]. The studies described herein clearly show that the DsbA-DsbB protein system is an attractive and tractable target for novel antibiotic development. While the inhibitors described above exhibit relatively weak binding affinities, the resulting phenotypes observed support their disruption of disulfide-bond formation in the cell. These "first-generation" molecules can serve as a foundation from which more potent compounds can be identified and developed.

**6. Conclusions**

humankind.

**Author details**

Bradley J. Landgraf1

**References**

, Guoping Ren1

1 New England Biolabs / Ruhr-Universität Bochum, Germany

\*Address all correspondence to: berkmen@neb.com

Struct. Mol. Biol. 11: 1179-85.

While more than 20 years of research have elucidated many of the Dsb proteins and their functions, more questions surrounding these proteins remain to be answered: What are the precise mechanisms by which PDI and DsbC catalyze disulfide-bond isomerization *in vivo*? How are electrons transported across the inner membrane by DsbD? What are the redox states and midpoint potentials of the cytoplasm of Crenarchaeota? Additionally, most of the characterization of Dsb proteins has been done in *E. coli*, which is not an appropriate model for all bacteria, e.g., *M. tuberculosis, Staphylococcus aureus,* and *Listeria monocytogenes*, so further characterization of the Dsb protein networks in other organisms is needed. Along these lines, Dsb proteins from pathogenic bacteria represent possible targets for antibiotic/vaccine development. Since several Dsb proteins have been structurally characterized, it is now possible to develop antibiotics by structure-guided design. While broad-spectrum antibiotic molecules are unlikely to be developed, again due to the diversity of Dsb proteins/networks within bac-

From Biology to Biotechnology: Disulfide Bond Formation in *Escherichia coli*

http://dx.doi.org/10.5772/67393

373

As more disulfide-bonded proteins are characterized, our knowledge of the stability and structures these bonds confer, their likelihood of scrambling in mulitply disulfide-bonded proteins, and their relative redox potentials will grow. This will allow researchers to better predict native disulfide bonds from sequence data and better engineer disulfide bonds in proteins for desirable physicochemical properties, which will benefit both the biotechnological and pharmaceutical industries, especially in the development and production of antibodies. Ideally, both industries should aim to produce antibodies as quickly, cheaply, and effectively as possible. The engineering of bacterial strains to overproduce correctly folded antibodies and/or engineering antibodies themselves for desired properties represents a technically challenging but incredibly useful advancement in the field of oxidative protein folding. Future research in these areas should lead to great innovations in both the biotechnological and pharmaceutical industries that will improve the health and increase the knowledge of

, Thorsten Masuch1

2 Department of Microbiology and Immunology, Harvard Medical School, Boston, MA, USA

[1] Lee C, Lee SM, Mukhopadhyay P, Kim SJ, Lee SC, et al. 2004. Redox regulation of OxyR requires specific disulfide bond formation involving a rapid kinetic reaction path. Nat.

, Dana Boyd2

and Mehmet Berkmen1

\*

terial species, those targeting specific pathogenic species are not out of reach.
