Site-directed mutagenesis provides insight into racemization and transamination of alanine catalyzed by Treponema denticola cystalysin

Barbara Cellini, Mariarita Bertoldi, Alessandro Paiardini, Simona D'Aguanno, Carla Borri Voltattorni

Research output: Contribution to journalArticle

Abstract

In addition to α, β-elimination of L-cysteine, Treponema denticola cystalysin catalyzes the racemization of both enantiomers of alanine accompanied by an overall transamination. Lys-238 and Tyr-123 or a water molecule located on the si and re face of the cofactor, respectively, have been proposed to act as the acid/base catalysts in the proton abstraction/donation at Cα/C4′ of the external aldimine. In this investigation, two site-directed mutants, K238A and Y123F, have been characterized. The Lys → Ala mutation results in the complete loss of either lyase activity or racemase activity in both directions or transaminase activity toward L-alanine. However, the K238A mutant is able to catalyze the overall transamination of D-alanine, and only D-alanine is the product of the reverse transamination. For Y123F the kcat/Km is reduced 3.5-fold for α,β- elimination, whereas it is reduced 300-400-fold for racemization. Y123F has ∼18% of wild type transaminase activity with L-alanine and an extremely low transaminase activity with D-alanine. Moreover, the catalytic properties of the Y124F and Y123F/Y124F mutants rule out the possibility that the residual racemase and transaminase activities displayed by Y123F are due to Tyr-124. All these data, together with computational results, indicate a two-base racemization mechanism for cystalysin in which Lys-238 has been unequivocally identified as the catalyst acting on the si face of the cofactor. Moreover, this study highlights the importance of the interaction of Tyr-123 with water molecules for efficient proton abstraction/donation function on the re face.

Original languageEnglish
Pages (from-to)36898-36905
Number of pages8
JournalJournal of Biological Chemistry
Volume279
Issue number35
DOIs
Publication statusPublished - Aug 27 2004

ASJC Scopus subject areas

  • Biochemistry

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