Development of novel bacterial strains for expressing properly folded and disulfide-bonded recombinant proteins; microbial Vitamin K epoxide reductase (VKOR) as a potential target for antibiotic development.

Protein therapeutics are an increasingly important class of compounds for clinical use. The integrity and reproducibility of the protein product, including proper folding and disulfide bond formation, are crucial aspects of the manufacturing process. Dr. Beckwith’s illuminating studies on disulfide bond formation have important ramifications for the expression of recombinant proteins in bacteria and eukaryotic host cells. For example, engineering cells that employ alternate ways of catalyzing disulfide bridges may have benefits in terms of efficiency and yield of recombinant protein expression, resulting in significant cost savings.

The discovery that coumarin-related anticoagulant compunds such as warfarin inhibit VKOR and are bactericidal support the notion that microbial VKOR is a validated and druggable target. The Beckwith lab is interested in small molecules that preferentially inhibit VKOR.
 

Dr. Beckwith’s lab employs E. coli as a model organism for tackling basic molecular mechanisms that are common to most cellular organisms.

The lab is studying the post-translational formation of disulfide bonds, an important component of the protein maturation process. In E. coli, this periplasmic-localized activity requires DsbA and membrane-embedded DsbB. However, alternative pathways can provide this function, and the lab is studying these redundant pathways. The lab’s recent findings relating to the bacterial version of VKOR and its ability to substitute for DsbB, raises intriguing questions about the function of eukaryotic VKOR. The laboratory is modifying certain enzymes to reconstitute disulfide bond capability in the cytoplasm of E. coli, and has successfully produced a fully functional eukaryotic protein that contains a complex disulfide bond network. 

There are two important elements for recycling protein functions: reduction of protein disulfide bonds in the cytoplasm; and the essential enzyme ribonucleotide reductase, that goes through a redox cycle involving cysteine to reduce ribonucleotides. The lab identified a mutant form of the peroxiredoxin AhpC that possesses disulfide reductase activity. It employed a genetic ablation of known biochemical pathways that generate reducing power, coupled with a screening for genetic mutations that can compensate for the non-functional genes and consequently restore cell viability. The lab is now trying to modify this AhpC moiety to increase the spectrum of cytoplasmic substrates that can be rescued when other disulfide bond reducing pathways are inactivated.
 

Dr. Beckwith has made landmark discoveries in the field of molecular biology, including the first isolation of a gene. He has received numerous awards, including the Abbott-ASM Lifetime Achievement Award from the American Society of Microbiology, and election to the prestigious National Academy of Sciences. His recent studies focused on diverse areas of molecular biology, including protein structure and shuttling that employ Escherichia coli (E. coli) as a model system. One area of particular interest relates to disulfide bond reduction, which is especially relevant to the ribonucleotide reductases family. These proteins rely on cysteine residues to drive the reduction of ribonucleotides, using a redox cycling. Dr. Beckwith’s lab defined the enzymatic requirements for the redox cycling, and uncovered compensatory mechanisms that operate when functionally inactivating mutations are introduced into protein components of the redox system. 

The lab has studied the enzymatic mechanisms responsible for protein disulfide bond formation in the periplasmic space, as well as evolutionary conserved aspects of this pathway. In a recent bioinformatic study that encompassed hundreds of bacterial species, Dr. Beckwith’s lab unexpectedly found that cysteine oxidation may not be a universal occurrence. Moreover, the lab discovered that some bacteria employ a functional equivalent of the E. Coli DsbB protein to generate disulfide bonds. The lab identified this enzyme as the bacterial ortholog of eukaryotic vitamin K epoxide reductase (VKOR) that is essential for blood clotting because it reduces vitamin K. This discovery is noteworthy because the C1 subunit of eukaryotic VKOR is the molecular target of the highly prescribed anticoagulant warfarin (brand name Coumadin). 

The lab also investigated bacterial cell division, which entails the congregation of multiple proteins into an aggregate designated as the divisome. The lab has developed a E. coli Artificial Septal Targeting technology that combines a bait and prey approach,with fluorescence microscopy, to tease apart stable protein-protein interactions that bolster the divisome. 
 

Biological Mechanisms and Pathways

• Bacteriology •
• Blood coagulation •
• Disulfide bond •
• Disulfide reductase •
• Electron transporter •
• Protein folding •
• Protein secretion •
• Protein structure •
• Redox cycle •
• Ribonucleotide reductase •
• Thioredoxin •
• Vitamin K epoxide reductase
•  

Infectious Disease

• Escherichia coli (E. coli)
•  

Research Tools and Instrumentation

• Escherichia coli (E. coli) •
• Heterologous protein expression
•  

Therapeutics

• Anticoagulant
•