Holo- and apo-cystalysin from Treponema denticola: two different conformations.
Cystalysin, the key virulence factor in the bacterium Treponema denticola responsible for periodontis, is a pyridoxal 5'-phosphate (PLP) enzyme which catalyzes, in addition to alpha,beta-elimination of L-cysteine, racemization and transamination of both enantiomers of alanine. In this paper several indicators have been used as probes of the different conformational status of T. denticola cystalysin in the holo and apo form. Compared to holoenzyme, the apoenzyme displays an altered reactivity of cysteine residues, a significantly different pI, and a differential susceptibility to proteinase K. The site of cleavage that is accessible in apocystalysin and masked in holocystalysin has been identified by mass spectrometry as the peptide bond between Phe 360 and Gly 361. This cleavage results in the loss of the C-terminal fragment corresponding to a molecular mass of 4289.21+/-0.1Da. The major fragment of cleaved enzyme retains its dimeric structure, binds the coenzyme with an affinity approximately 5000-fold lower than that of uncleaved holoenzyme, and in the reconstituted form is able to form the external aldimine with substrates. Although the break causes the loss of lyase, racemase and transaminase activities of D-alanine, it does not abolish the transaminase activity of L-alanine. Possible mechanistic and physiological implications are proposed.
Cellini
Barbara
B
Dipartimento di Scienze Morfologico-Biomediche, Sezione di Chimica Biologica, Facoltà di Medicina e Chirurgia, Strada Le Grazie 8, Universita di Verona, 37134 Verona, Italy.
Montioli
Riccardo
R
Bossi
Alessandra
A
Bertoldi
Mariarita
M
Laurents
Douglas V
DV
Voltattorni
Carla Borri
CB
D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D002942:Circular Dichroism; D003542:Cystathionine gamma-Lyase / Q000737:chemistry* / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K / Q000378:metabolism; D004789:Enzyme Activation; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D007525:Isoelectric Focusing; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D048373:Treponema denticola / Q000201:enzymology*
D001051:Apoenzymes; D020035:Holoenzymes; D019286:Endopeptidase K; C400145:cystalysin; D003542:Cystathionine gamma-Lyase; D003545:Cysteine