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 Journal Article
| Authors: | Uson, M. L.; Carl, A.; Goldgur, Y.; Shuman, S. |
| Article Title: | 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 |
| 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 -loop that coordinates a fourth manganese ion (M4). © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research. |
| Journal Title: | Nucleic Acids Research |
| Volume: | 46 |
| Issue: | 8 |
| ISSN: | 0305-1048 |
| Publisher: | Oxford University Press |
| Date Published: | 2018-05-01 |
| Start Page: | 4164 |
| End Page: | 4175 |
| Language: | English |
| DOI: | 10.1093/nar/gky238 |
| PROVIDER: | scopus |
| PMCID: | PMC5934675 |
| PUBMED: | 29635474 |
| DOI/URL: | |
| Notes: | Article -- Export Date: 1 October 2018 -- Source: Scopus |
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