Parvulin

The smallest member of a new family of prolyl iso-merases (unrelated to the cyclophilins or the FK-506 binding proteins) that catalyzes the proline-limited folding of a variant of ribonuclease T1 with a KJK value of 30,000 M s With the tetrapeptide succinyl-Ala-Leu-Pro-Phe-4-nitroanilide as a substrate in parvulin-catalyzed prolyl isomerization, this parameter is 1.1 x 10 M s Parvulin also accelerates its own refolding in an autocatalytic fashion. 

The third family of PPIases, the parvulins, does not belong to immunophilins because family members do not show affinity to immunosuppressive drugs. In all cases the prototypic family members are small, single-domain proteins expressed in high abundance predominantly in the cytosol of mammalian and bacterial cells. 

Fig. 10.3 Human parvulins. Protein nomenclature is according to Fischer [3], The gene name and the molecular mass of the unprocessed proteins are shown in the brackets. In the second row, the accession number ofthe SWISS-PROT/ TREM BL database and an example of an alternative name ofthe protein is given. The amino acid residues that border the protein domains or functional modules are designated according to SWISS-PROT or Pfam databases. The parvulin domain is depicted in yellow. Signal sequence regions are shown as colorless boxes. WW, WW domain.

Two conserved signature sequences containing histidine motifs (His-Xaa-Val(Ile)-Xaa-Lys and Gly-Xaa-His-Ile(Leu,Val)-Ile), which are separated by a stretch of about 70-85 amino acids containing a few other conserved residues, characterize the catalytic core of all parvulins. In various plants, one member corresponds... 

Although the X-ray structure of a Pinl-dipeptide complex implicated covalent catalysis, a lack of nucleophilic assistance in the catalytic pathway can be derived from the covalent modification of parvulins by 5-OH-l,4-naphthoquinone (juglone). A juglone-inactivated E. coli ParlO contains two inhibitor molecules that are covalently bound to the side-chains of Cys41 and Cys69 because of a Michael... 

Interestingly, FKBP12 does not show diffusion control of kc J Km, and proton inventory does not show a bulging down curve. This case underlines the idea that a distinct catalytic mechanism exists for FKBP compared with cydophilins and parvulins. 

The PPIase domains of cyclophilin and FKBPs do not display any similarities whereas members within each subfamily show a high degree of homology in sequence and in three-dimensional structure, suggesting that conservation of the overall shape of the active site and of certain residues is essential for PPIase activity. Moreover, both enzymes feature structural differences from parvulins, in particular Pinl (Fig. 12.2) [1,9,12]. 

Other parvulins also give important structural and mechanistic information. In particular, PinlAt, the Pinl homolog from Arabidopsis thaliana, displays a high degree of similarity with Pinl despite the absence of the Trp-Trp site. In particular, all residues defined as essential in Pinl are represented in PinlAt. In this enzyme, however, binding of the substrate seems to cause the Cys70 side-chain to protrude outside the catalytic site, making it unlikely to interact with the amino acyl proline peptide bond [157]. 

Prolyl isomerases belong to three structurally diverse families the cyclophilins, the FK506 binding proteins, and the parvulins. Usually, they are abbreviated to Cyp, FKBP and Parv, followed by the molecular mass and, sometimes, an indicator for the subcellular localization. Cypl8cy thus denotes the cytoplasmic cyclophilin with a molecular mass of 18 kDa. This cyclophilin is also often referred to as CypA. 

Several of the proteins with parvulin domains seem to be involved in protein maturation, particularly of secreted proteins, and it has been suggested that mutations cause misfolding and defects in the function of other proteins. The SurA protein is located in the periplasm of E. coli and participates in early stages of the maturation of proteins of the outer membrane (Lazar and Kolter, 1996 Rouviere and Gross, 1996 Wild et al., 1996). SurA is also involved in the pathway that signals the accumulation of unfolded proteins from the periplasm to the cytoplasm (Missiakas and Raina, 1997). Pinl is an essential regulator protein of the cell cycle (Hunter, 1998). The possible functions of SurA and Pinl are further discussed in Section XI. 

The prolyl isomerases catalyze isomerizations only at prolyl bonds and not at nonprolyl peptide bonds. The refolding of the P39A variant of RNase Tl, which is limited in rate by the very slow trans —> cis reisomerization of the Tyr38-Ala39 bond (see Section IV.B), is not catalyzed by cyclophilins, FKBPs, or parvulins. These enzymes are also unable to catalyze amide bond isomerizations in the proline-free model peptide Ala-Ala-Tyr-Ala-Ala (Scholz etal., 1998b). 

The periplasm of E. coli contains prolyl isomerases of all three families. PPIA is a member of the cyclophilin family (Liu and Walsh, 1990 Hayano et al., 1991), FkpA or FKBP26 (Horne and Young, 1995) contains a FKBP12-like domain, and SurA (Rouviere and Gross, 1996) and PpiD (Dartigalongue and Raina, 1998) show parvulin domains. SurA is a soluble protein, whereas PpiD is attached to the inner membrane with its catalytic domain exposed to the periplasmic space. 

Behrens and colleagues (2001) investigated SurA both in vivo and in vitro. All regions of this large protein contribute to its function in vivo. Prolyl isomerase activity could be demonstrated for one of the two parvulin domains, but, as in the case of FkpA, this activity seems to be unimportant for SurA function in vivo. SurA has a chaperone function in vitro, which requires the N-terminal part of the protein to be present. 

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