In Vivo Formation of the Protein Disulfide Bond That Enhances the Thermostability of Diphosphomevalonate Decarboxylase, an Intracellular Enzyme from the Hyperthermophilic Archaeon Sulfolobus solfataricus

Abstract

In the present study, the crystal structure of recombinant diphosphomevalonate decarboxylase from the hyperthermophilic archaeon Sulfolobus solfataricus was solved as the first example of an archaeal and thermophile-derived diphosphomevalonate decarboxylase. The enzyme forms a homodimer, as expected for most eukaryotic and bacterial orthologs. Interestingly, the subunits of the homodimer are connected via an intersubunit disulfide bond, which presumably formed during the purification process of the recombinant enzyme expressed in Escherichia coli. When mutagenesis replaced the disulfide-forming cysteine residue with serine, however, the thermostability of the enzyme was significantly lowered. In the presence of ␤-mercaptoethanol at a concentration where the disulfide bond was completely reduced, the wild-type enzyme was less stable to heat. Moreover, Western blot analysis combined with nonreducing SDS-PAGE of the whole cells of S. solfataricus proved that the disulfide bond was predominantly formed in the cells. These results suggest that the disulfide bond is required for the cytosolic enzyme to acquire further thermostability and to exert activity at the growth temperature of S. solfataricus. IMPORTANCEThis study is the first report to describe the crystal structures of archaeal diphosphomevalonate decarboxylase, an enzyme involved in the classical mevalonate pathway. A stability-conferring intersubunit disulfide bond is a remarkable feature that is not found in eukaryotic and bacterial orthologs. The evidence that the disulfide bond also is formed in S. solfataricus cells suggests its physiological importance.

A Single Amino Acid Mutation Converts (R)-5-Diphosphomevalonate Decarboxylase into a Kinase

“…X-ray Crystallographic Analysis of the Complex Structures of SsoDMD-Recombinant expression and purification for the crystallization of SsoDMD were performed as described in our previous study (17). The purified recombinant SsoDMD was crystallized at 20°C using the hanging-drop vapor diffusion method.…”

“…3, A and B). A disulfide-bonded dimer is the expected physiological quaternary structure of SsoDMD (17). In the crystals, three dimers are related to a 3-fold crystallographic symmetry axis, although there is one monomer in the asymmetric unit.…”

“…Substrates-We recently have reported the X-ray crystallographic structures of substrate-free SsoDMD (17). Here, we succeeded in solving the structures of SsoDMD in complex with ATP␥S and MVA-5-PP (the complex SsoDMD⅐ ATP␥S⅐MVA-5-PP) at a 1.5 Å resolution, and with ADP and MVA-5-PP (SsoDMD⅐ADP⅐MVA-5-PP) at a 1.7 Å resolution.…”

“…The crystal structures of DMD have been solved with eukaryotic enzymes from Saccharomyces cerevisiae (12), Trypanosoma brucei (13), mice, and humans (14); with bacterial enzymes from Staphylococcus aureus (13), Streptococcus pyogenes, Legionella pneumophila, and Staphylococcus epidermidis (15,16); and with an archaeal enzyme from Sulfolobus solfataricus (17). Among them, only those of DMD from S. epidermidis were solved in complex forms with the substrates or inhibitors ( Fig.…”

Lipid sugar carriers at the extremes: The phosphodolichols Archaea use in N-glycosylation

“…Here too, Archaea have introduced unusual variations not seen elsewhere. For instance, while the mevalonate pyrophosphate decarboxylase in S. solfataricus exists as a dimer, like its eukaryal and bacterial counterparts, the archaeal enzyme is unique in that a disulfide bond serves to stabilize the dimer in this thermoacidophile [78]. Recently, evidence for the existence of yet another pathway for IPP biosynthesis in Archaea has been presented.…”