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Nucleic Acids Res. 2018 May 4;46(8):4164-4175. doi: 10.1093/nar/gky238.

Crystal structure and mutational analysis of Mycobacterium smegmatis FenA highlight active site amino acids and three metal ions essential for flap endonuclease and 5' exonuclease activities.

Author information

1
Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.
2
Structural Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.

Abstract

Mycobacterium smegmatis FenA is a nucleic acid phosphodiesterase with flap endonuclease and 5' exonuclease activities. The 1.8 Å crystal structure of FenA reported here highlights as its closest homologs bacterial FEN-family enzymes ExoIX, the Pol1 exonuclease domain and phage T5 Fen. Mycobacterial FenA assimilates three active site manganese ions (M1, M2, M3) that are coordinated, directly and via waters, to a constellation of eight carboxylate side chains. We find via mutagenesis that the carboxylate contacts to all three manganese ions are essential for FenA's activities. Structures of nuclease-dead FenA mutants D125N, D148N and D208N reveal how they fail to bind one of the three active site Mn2+ ions, in a distinctive fashion for each Asn change. The structure of FenA D208N with a phosphate anion engaged by M1 and M2 in a state mimetic of a product complex suggests a mechanism for metal-catalyzed phosphodiester hydrolysis similar to that proposed for human Exo1. A distinctive feature of FenA is that it does not have the helical arch module found in many other FEN/FEN-like enzymes. Instead, this segment of FenA adopts a unique structure comprising a short 310 helix and surface β-loop that coordinates a fourth manganese ion (M4).

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