Distinct enzymatic strategies for de novo generation of disulfide bonds in membranes

Abstract

Disulfide bond formation is a catalyzed reaction essential for the folding and stability of proteins in the secretory pathway. In prokaryotes, disulfide bonds are generated by DsbB or VKOR homologs that couple the oxidation of a cysteine pair to quinone reduction. Vertebrate VKOR and VKOR-like enzymes have gained the epoxide reductase activity to support blood coagulation. The core structures of DsbB and VKOR variants share the architecture of a four-transmembrane-helix bundle that supports the coupled redox reaction and a flexible region containing another cysteine pair for electron transfer. Despite considerable similarities, recent high-resolution crystal structures of DsbB and VKOR variants reveal significant differences. DsbB activates the cysteine thiolate by a catalytic triad of polar residues, a reminiscent of classical cysteine/serine proteases. In contrast, bacterial VKOR homologs create a hydrophobic pocket to activate the cysteine thiolate. Vertebrate VKOR and VKOR-like maintain this hydrophobic pocket and further evolved two strong hydrogen bonds to stabilize the reaction intermediates and increase the quinone redox potential. These hydrogen bonds are critical to overcome the higher energy barrier required for epoxide reduction. The electron transfer process of DsbB and VKOR variants uses slow and fast pathways, but their relative contribution may be different in prokaryotic and eukaryotic cells. The quinone is a tightly bound cofactor in DsbB and bacterial VKOR homologs, whereas vertebrate VKOR variants use transient substrate binding to trigger the electron transfer in the slow pathway. Overall, the catalytic mechanisms of DsbB and VKOR variants have fundamental differences.

Keywords:

• Disulfide bond
• integral membrane enzyme
• DsbB
• VKOR
• quinone
• vitamin K

Disclosure statement

No potential conflict of interest was reported by the author.

Additional information

Funding

W.L. is supported by NHLBI [R01 HL121718], W. M. Keck Foundation (Forefront of Science Award), American Heart Association [Collaboration Science Award 20CSA35310354 and Established Investigator Award 23EIA1039588], Children’s Discovery Institute [MCII 2020-854], NIAID [R01 AI158500], and NIGMS [R01 GM131008].