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

Abstract

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 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 β-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 Leu428, 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 Leu428 deletion mutant for L-proline was about 800 times larger than the Km value of the wild-type enzyme, although the kcat values did not differ much between the two enzymes. This suggests the C-terminal Leu428 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. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.