Naslov
Asp214→Ala Mutation Reorganizes the Active Site of Citrobacter freundii Tyrosine Phenol-lyase

Autori
Milić, Dalibor ; Demidkina, Tatyana V. ; Matković-Čalogović, Dubravka ; Antson, Alfred A.

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
26th European Crystallographic Meeting, ECM 26, Darmstadt, 2010 Acta Crystallographica Section A - Foundations of Crystallography, vol. 66 / - , 2010, S280-s281

Skup
26th European Crystallographic Meeting

Mjesto i datum
Darmstadt, Njemačka, 29.08.2010. - 02.09.2010

Vrsta sudjelovanja
Poster

Vrsta recenzije
Nije recenziran

Ključne riječi
vitamin B6; enzymatic structure-activity relationships; mutations

Sažetak
Tyrosine phenol-lyase (TPL) from Citrobacter freundii and other bacteria is a homotetrameric protein. This pyridoxal-5'-phosphate (PLP)-dependent enzyme catalyzes the reversible hydrolytic cleavage (β-elimination reaction) of L-tyrosine to give phenol and ammonium pyruvate [1]. The active site (one per each subunit) is situated at an interface between the small and large domain of one subunit, and the large domain of the adjacent subunit [2]. It can assume either the open or closed conformation [3]. We suggest that the active site closure is fundamental to the cleavage of L-tyrosine Cβ–Cγ bond [4]. D214A and D214N TPL mutants from C. freundii were shown to be inactive for the β-elimination of L-tyrosine and its derivatives [5]. We present the structure of D214A TPL mutant solved at 1.9 Å resolution. This structure indicates that the mutation prevents the protonated N1 nitrogen atom of the PLP pyridine ring from creating a hydrogen-bond with the side chain of residue 214. Instead, new hydrogen bonds are formed between the PLP O3' atom and the side chains of Asn185 and Thr216, and between the deprotonated PLP N1 atom and a nearby water molecule that is hydrogen bonded with Glu103 and Thr126. These new interactions cause a significant conformational change in the active site, including reorientation of PLP and the side chains of Asn185 and Glu103, and translocation of the helix α4', together with the catalytically important Thr124, by ~1.5 Å from the active site cleft. The open active site cleft as a whole is slightly more closed than in the wild type holoenzyme. These results explain the observed inactivity of D214A mutant [5]. [1] Phillips R.S. ; Demidkina, T.V. ; Faleev, N.G. Biochim. Biophys. Acta, 2003, 1647, 167. [2] Antson, A.A. ; Demidkina, T.V. ; Gollnick, P. ; Dauter, Z. ; Von Tersch, R.L. ; Long, J. ; Berezhnoy, S.N. ; Phillips, R.S. ; Harutyunyan, E.H. ; Wilson, K.S. Biochemistry, 1993, 32, 4195. [3] Milić, D. ; Matković-Čalogović, D. ; Demidkina, T.V. ; Kulikova, V.V. ; Sinitzina, N.I. ; Antson, A.A. Biochemistry, 2006, 45, 7544. [4] Milić, D. ; Demidkina, T.V. ; Faleev, N.G. ; Matković-Čalogović, D. ; Antson, A.A. J. Biol. Chem. 2008, 283, 29, 29206. [5] Demidkina, T.V. ; Faleev, N.G. ; Papisova, A.I. ; Bazhulina, N.P. ; Kulikova, V.V. ; Gollnick, P.D. ; Phillips, R.S. Biochim. Biophys. Acta, 2006, 1764, 1268.

Izvorni jezik
Engleski

Znanstvena područja
Kemija

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