Holo-and apocystalysin 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 α,β-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 ± 0.1 Da. The major fragment of cleaved enzyme retains its dimeric structure, binds the coenzyme with an affinity ∼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. © 2006 Elsevier Inc. All rights reserved.