The lon protease from Mycobacterium smegmatis: molecular cloning, sequence analysis, functional expression, and enzymatic characterization

Biochemistry
S G RoudiakT E Shrader

Abstract

We have charterized a Mycobacterium smegmatis gene encoding a homolog of the ATP-dependent protease Lon (La). Our identification of a Lon homolog, in conjunction with our previous work, identifies M. smegmatis as the first known example of a eubacterium containing both Lon and a complete 20S proteasome (containing both alpha- and beta-subunits). Despite the significant primary sequence divergence between M. smegmatis Lon (Ms-Lon) and E. coli Lon (Ec-Lon), expression of Ms-Lon was only moderately toxic to E. coli cells. The ability of E. coli cells to tolerate expression of Ms-Lon reveals that Ms-Lon does not recognize and degrade essential E. coli proteins. We conclude that discrimination against nonsubstrate proteins is broadly conserved between Ec-Lon and Ms-Lon. Additional conservation of substrate recognition was demonstrated by the ability of Ms-Lon to degrade efficiently RcsA, a natural substrate of Ec-Lon. Purified Ms-Lon displays chymotrypsin-like specificity in peptidase assays that are stimulated by unfolded protein and supported by nonhydrolyzed nucleotide analogs. Maximal peptidase activity requires ATP or dATP. Replacement of Ms-Lon's catalytic Ser with Ala (S675A), Thr (S675T), or Cys (S675C) reduced to background...Continue Reading

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Citations

Mar 19, 1999·Trends in Microbiology·R De MotW Baumeister
May 14, 1999·Current Opinion in Microbiology·S Gottesman
May 16, 2013·Proceedings of the National Academy of Sciences of the United States of America·Ellen F VieuxTania A Baker
Jun 27, 2012·The Journal of Biological Chemistry·Magdalena BartoszewskaIda J van der Klei
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May 26, 1999·Proceedings of the National Academy of Sciences of the United States of America·L Van Melderen, S Gottesman
Jan 13, 2016·Scientific Reports·Yuan Lu, James R Swartz
Aug 1, 2006·Protein Science : a Publication of the Protein Society·Tatyana V RotanovaAlexander Wlodawer
Sep 16, 2011·The Journal of Antimicrobial Chemotherapy·Rashmi TandonMridula Bose
Oct 10, 1998·Journal of Bacteriology·I NagyR De Mot

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