Figure 4.

Lart1 targets a conserved arginine residue in the NAD+-binding pocket of glutamate dehydrogenase.A, fragmentation pattern of Gdh2p peptide containing phosphoribosyl-Arg800 identified by LC-MS/MS. The precursor [M+3H]³⁺ ion, m/z 657.30, is labeled with an asterisk (∗) and was subjected to HCD fragmentation to generate the spectrum shown. The modification site was localized to arginine 800 highlighted in red. The b10 fragment ion containing the modified residue shows neutral loss of the phosphoribosyl group (−212 Da). B, endpoint assays depicting incorporation of [³²P]-NAD+ by WT Lart1 into the indicated alanine mutants of Gdh2p. Mutation of the ADPR acceptor site R800 abolishes ADP-ribosylation. C, model of DdGlud2 built by Phyre (64) using Pyrococcus furiosus GDH (PDB 1HRD) as a template, indicating the position of the ADP-ribose acceptor residue Arg 763 (rendered in sticks). Bound NADH and glutamate (Glu), (modeled by alignment to liganded bovine GDH, PDB 6DHQ) are rendered in sticks. D, sequence logos depicting the conservation of R800 and the surrounding residues in GDH enzymes from fungi (top, based on MAFFT alignment of 125 sequences), amoeba (middle, based on MAFFT alignment of 19 sequences), and metazoans (bottom, based on MAFFT alignment of 145 sequences). Red arrows indicate the position of the arginine targeted by Lart1. LC-MS/MS, liquid chromatography/mass spectrometry.