Mycobacterium tuberculosis prcBA genes encode a gated proteasome with broad oligopeptide specificity.
Genes predicted to be associated with the putative proteasome of Mycobacterium tuberculosis (Mtb) play a critical role in defence of the bacillus against nitrosative stress. However, proteasomes are uncommon in eubacteria and it remains to be established whether Mtb's prcBA genes in fact encode a proteasome. We found that coexpression of recombinant PrcB and PrcA in Escherichia coli over a prolonged period at 37 degrees C allowed formation of an alpha(7)beta(7)beta(7)alpha(7), 750 kDa cylindrical stack of four rings in which all 14 beta-subunits were proteolytically processed to expose the active site threonine. In contrast to another Actinomycete, Rhodococcus erythropolis, Mtb's beta-chain propeptide was not required for particle assembly. Peptidolytic activity of the 750 kDa particle towards a hydrophobic oligopeptide was nearly two orders of magnitude less than that of the Rhodococcus 20S proteasome, and unlike eukaryotic and archaeal proteasomes, activity of the Mtb 750 kDa particle could not be stimulated by SDS, Mg(2+) or Ca(2+). Electron microscopy revealed what appeared to be obstructed alpha-rings in the Mtb 750 kDa particle. Deletion of the N-terminal octapeptide from Mtb's alpha-chain led to disappearance of the apparent obstruction and a marked increase of peptidolytic activity. Unlike proteasomes isolated from other Actinomycetes, the open-gate Mtb mutant 750 kDa particle cleaved oligopeptides not only after hydrophobic residues but also after basic, acidic and small, neutral amino acids. Thus, Mtb encodes a broadly active, gated proteasome that may work in concert with an endogenous activator.
Lin
Gang
G
Department of Microbiology and Immunology, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA.
Hu
Guiqing
G
Tsu
Christopher
C
Kunes
Yune Z
YZ
Li
Huilin
H
Dick
Lawrence
L
Parsons
Thomas
T
Li
Ping
P
Chen
Zhiqiang
Z
Zwickl
Peter
P
Weich
Nadine
N
Nathan
Carl
C
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D015964:Gene Expression Regulation, Bacterial; D015640:Ion Channel Gating; D008854:Microscopy, Electron; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D061988:Proteasome Inhibitors; D013379:Substrate Specificity; D013381:Subtilisins / Q000235:genetics* / Q000378:metabolism; D017186:Titrimetry
D001426:Bacterial Proteins; D009842:Oligopeptides; C102527:PrcA protein, bacteria; D011480:Protease Inhibitors; D061988:Proteasome Inhibitors; D013381:Subtilisins; D046988:Proteasome Endopeptidase Complex