Supporting Information
Role of Active-Site Residues Tyr55 and Tyr114 in Catalysis and Substrate Specificity of
Corynebacterium diphtheriae C-S Lyase
Alessandra Astegno*, Alessandra Allegrini, Stefano Piccoli, Alejandro Giorgetti, and Paola Dominici
Department of Biotechnology, University of Verona, Strada Le Grazie 15, Verona (VR), Italy.
*To whom correspondence should be addressed:
Alessandra Astegno, Department of Biotechnology, University of Verona, Strada Le Grazie 15, Verona
(VR), Italy.
Tel: +39 045 8027955; Fax: +39 045 8027929;
e-mail: alessandra.astegno@univr.it
Supplementary Figures
Figure S1. RMSD vs. time plot for the simulation performed on C-S lyase from C. diphtheriae. The RMSD
was calculated on the backbone atoms and stabilized to ca. 0.43 nm, after 90 ns of simulation time.
Figure S2. Active site of C-S lyase from C. diphtheria. Binding of PLP-L-serine (PLP-SER) or PLP-Oacetyl-L-serine (PLP-OAS), to the wild type (panels A, C, respectively) and to Y114F protein (panel B, D,
respectively). Chain A of the protein is represented in pink and chain B is shown in green. Tyr55, Tyr114,
Lys222, and Arg351 are depicted in dark green and represented in licorice. Ligands are depicted in gold and
represented in a ball and stick model. Hydrogen atoms are colored in cyan. While the formation of a
hydrogen bond between the hydroxyl group of Tyr114 and the hydroxyl group of the PLP-L-serine can be
noted in the wild type enzyme (panel A, the black dotted line emphasizes the hydrogen bond), this
interaction is lost in the Y114F mutant (panel B). While the methyl group of PLP-O-acetyl-L-serine cannot
interact with Tyr114 (panel C), the Y114F mutation allows formation of hydrophobic interactions between
the aromatic ring of Phe114 and the methyl group of PLP-O-acetyl-L-serine (panel D).
Supplementary Methods
RMSD calculation
The RMSD graph was obtained using Gromacs (1) g_rms tool.
Active-site modelling
The structure of PLP-L-serine was obtained using Chimera software (2) and superimposed to the PLP-Lcystathionine coordinates of C-S lyase structure from C. diphtheriae described in the main article.
Superposition of structures was carried out using Chimera software. The mutated variant of C-S lyase
carrying a Phe at position 114 was obtained using the Chimera program (2). The same protocol was used for
the creation of C-S lyase binding PLP-O-acetyl-L-serine, in the wild type and the Y114F mutant forms.
Energy minimization stage on C-S lyase structures was performed using the Gromacs program (1), following
the same protocol described in the main article. Parameters for PLP-L-serine and PLP-O-acetyl-L-serine
ligands were derived using the PRODRG server (3). This protocol has already been used successfully by us
(4-6). Energy convergence was reached at 100,000 steps.
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