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Crystal structure of novel dye-linked L-proline dehydrogenase from hyperthermophilic archaeon Aeropyrum pernix.

ABSTRACT: Two types of dye-linked L-proline dehydrogenase (PDH1, ?4?4-type hetero-octamer, and PDH2, ????-type heterotetramer) have been identified so far in hyperthermophilic archaea. Here, we report the crystal structure of a third type of L-proline dehydrogenase, found in the aerobic hyperthermophilic archaeon Aeropyrum pernix, whose structure (homodimer) is much simpler than those of previously studied L-proline dehydrogenases. The structure was determined at a resolution of 1.92 Å. The asymmetric unit contained one subunit, and a crystallographic 2-fold axis generated the functional dimer. The overall fold of the subunit showed similarity to that of the PDH1 ?-subunit, which is responsible for catalyzing L-proline dehydrogenation. However, the situation at the subunit-subunit interface of the A. pernix enzyme was totally different from that in PDH1. The presence of additional surface elements in the A. pernix enzyme contributes to a unique dimer association. Moreover, the C-terminal Leu(428), which is provided by a tail extending from the FAD-binding domain, shielded the active site, and an L-proline molecule was entrapped within the active site cavity. The K(m) value of a Leu(428) deletion mutant for L-proline was about 800 times larger than the K(m) value of the wild-type enzyme, although the k(cat) values did not differ much between the two enzymes. This suggests the C-terminal Leu(428) is not directly involved in catalysis, but it is essential for maintaining a high affinity for the substrate. This is the first description of an LPDH structure with L-proline bound, and it provides new insight into the substrate binding of LPDH.

SUBMITTER: Sakuraba H

PROVIDER: S-EPMC3370190 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Crystal structure of novel dye-linked L-proline dehydrogenase from hyperthermophilic archaeon Aeropyrum pernix.

Sakuraba Haruhiko H Satomura Takenori T Kawakami Ryushi R Kim Kwang K Hara Yusuke Y Yoneda Kazunari K Ohshima Toshihisa T

The Journal of biological chemistry 20120416 24

Two types of dye-linked L-proline dehydrogenase (PDH1, α4β4-type hetero-octamer, and PDH2, αβγδ-type heterotetramer) have been identified so far in hyperthermophilic archaea. Here, we report the crystal structure of a third type of L-proline dehydrogenase, found in the aerobic hyperthermophilic archaeon Aeropyrum pernix, whose structure (homodimer) is much simpler than those of previously studied L-proline dehydrogenases. The structure was determined at a resolution of 1.92 Å. The asymmetric uni ...[more]

PMID: 22511758

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Project description:Pernisine is a subtilisin-like protease that was originally identified in the hyperthermophilic archaeon Aeropyrum pernix, which lives in extreme marine environments. Pernisine shows exceptional stability and activity due to the high-temperature conditions experienced by A. pernix Pernisine is of interest for industrial purposes, as it is one of the few proteases that has demonstrated prion-degrading activity. Like other extracellular subtilisins, pernisine is synthesized in its inactive pro-form (pro-pernisine), which needs to undergo maturation to become proteolytically active. The maturation processes of mesophilic subtilisins have been investigated in detail; however, less is known about the maturation of their thermophilic homologs, such as pernisine. Here, we show that the structure of pro-pernisine is disordered in the absence of Ca2+ ions. In contrast to the mesophilic subtilisins, pro-pernisine requires Ca2+ ions to adopt the conformation suitable for its subsequent maturation. In addition to several Ca2+-binding sites that have been conserved from the thermostable Tk-subtilisin, pernisine has an additional insertion sequence with a Ca2+-binding motif. We demonstrate the importance of this insertion for efficient folding and stabilization of pernisine during its maturation. Moreover, analysis of the pernisine propeptide explains the high-temperature requirement for pro-pernisine maturation. Of note, the propeptide inhibits the pernisine catalytic domain more potently at high temperatures. After dissociation, the propeptide is destabilized at high temperatures only, which leads to its degradation and finally to pernisine activation. Our data provide new insights into and understanding of the thermostable subtilisin autoactivation mechanism.IMPORTANCE Enzymes from thermophilic organisms are of particular importance for use in industrial applications, due to their exceptional stability and activity. Pernisine, from the hyperthermophilic archaeon Aeropyrum pernix, is a proteolytic enzyme that can degrade infective prion proteins and thus has a potential use for disinfection of prion-contaminated surfaces. Like other subtilisin-like proteases, pernisine needs to mature through an autocatalytic process to become an active protease. In the present study, we address the maturation of pernisine and show that the process is regulated specifically at high temperatures by the propeptide. Furthermore, we demonstrate the importance of a unique Ca2+-binding insertion for stabilization of mature pernisine. Our results provide a novel understanding of thermostable subtilisin autoactivation, which might advance the development of these enzymes for commercial use.

Dataset Information

Crystal structure of novel dye-linked L-proline dehydrogenase from hyperthermophilic archaeon Aeropyrum pernix.

Publications

Crystal structure of novel dye-linked L-proline dehydrogenase from hyperthermophilic archaeon Aeropyrum pernix.

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