title: The lon protease from Mycobacterium smegmatis: molecular cloning, sequence analysis, functional expression, and enzymatic characterization.
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 levels Ms-Lon's in vitro peptidase activity. However, by employing a sensitive in vivo assay, based on the degradation of RcsA, we demonstrated that the S675C variant retained specific protease activity. Finally, variants of Ms-Lon, with substututions at or near S675, reduce the enzyme's basal ATPase activity, suggesting a structural interaction between the peptidase and ATPase active sites of Ms-Lon.
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
authors.forename: S G
authors.initials: SG
authors.lastname: Roudiak
authors.forename: A
authors.initials: A
authors.lastname: Seth
authors.forename: N
authors.initials: N
authors.lastname: Knipfer
authors.forename: T E
authors.initials: TE
authors.lastname: Shrader
mesh_terms: D049069:ATP-Dependent Proteases; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009161:Mycobacterium / Q000201:enzymology* / Q000235:genetics*; D049070:Protease La; D046988:Proteasome Endopeptidase Complex; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011742:Pyrimidine Nucleotides / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D013045:Species Specificity; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D009097:Multienzyme Complexes; D011742:Pyrimidine Nucleotides; D011993:Recombinant Fusion Proteins; D049069:ATP-Dependent Proteases; D012697:Serine Endopeptidases; C483567:Lon protein, E coli; D049070:Protease La; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000251:Adenosine Triphosphatases