Ssp1, a site-specific parvulin homolog from Neurospora crassa active in protein folding.

Peptidyl-prolyl cis-trans-isomerases (PPIases) are enzymes capable of isomerizing a Xaa-Pro peptide bond. Three families of PPIases are known: cyclophilins, FKBPs, and parvulins. The physiological functions of the PPIases are only poorly understood. Eucaryotic members of the parvulin family have recently been shown to be essential for regulation of mitosis. Here we describe the purification and characterization of Ssp1, an abundant parvulin homolog from Neurospora crassa, which is unique among the known eucaryotic parvulins in containing a polyglutamine stretch between the N-terminal WW domain and the C-terminal PPIase domain. Ssp1 is a site-specific PPIase with respect to the amino acid N-terminal to the proline residue. Peptides with glutamate, phosphoserine, or phosphothreonine in the -1-position proved to be the best substrates. Ssp1 is not only able to isomerize small peptides but is also active in protein folding, as shown with mouse dihydrofolate reductase. Using the substrate specificity of Ssp1, we could identify Glu81-Pro82 as a PPIase-sensitive site in folding of dihydrofolate reductase. These results demonstrate that Ssp1 is a potent mediator of protein folding and that parvulins can serve as tools to elucidate rate-limiting steps in protein folding reactions.

The Escherichia coli SlyD is a metal ion-regulated peptidyl-prolyl cis/trans-isomerase.

In Escherichia coli as many as nine different genes coding for proteins with significant homology to peptidyl-prolyl cis/trans-isomerases (PPIases) have been found. However, for three of them, the histidine-rich SlyD, the homologous gene product of ORF149, and parvulin-like SurA, it was not known whether these proteins really possess PPIase activity. To gain access to the full set of PPIases in E. coli, SlyD, the N-terminal fragment of SlyD devoid of the histidine-rich region, as well as the protein product of ORF149 of E. coli named SlpA (SlyD-like protein) were cloned, overexpressed, and purified to apparent homogeneity. On the basis of the amino acid sequences, both proteins proved to be of the FK506-binding protein type of PPIases. Only when using trypsin instead of chymotrypsin as helper enzyme in the PPIase assay, the enzymatic activity of full-length SlyD and its N-terminal fragment can be measured. For Suc-Ala-Phe-Pro-Arg-4-nitroanilide as substrate, kcat/Km of 29,600 M-1 s-1 for SlyD and 18,600 M-1 s-1 for the N-terminal fragment were obtained. Surprisingly, the PPIase activity of SlyD is reversibly regulated by binding of three Ni2+ ions to the histidine-rich, C-terminal region. Because the PPIase activity of SlpA could be established as well, we now know eight distinct PPIases with proven enzyme activity in E. coli.