title: Structural and functional assays of AtTLP18.3 identify its novel acid phosphatase activity in thylakoid lumen.
abstract: The membrane protein AtTLP18.3 of Arabidopsis (Arabidopsis thaliana) contains a domain of unknown function, DUF477; it forms a polysome with photosynthetic apparatuses in the thylakoid lumen. To explore the molecular function of AtTLP18.3, we resolved its crystal structures with residues 83 to 260, the DUF477 only, and performed a series of biochemical analyses to discover its function. The gene expression of AtTLP18.3 followed a circadian rhythm. X-ray crystallography revealed the folding of AtTLP18.3 as a three-layer sandwich with three α-helices in the upper layer, four β-sheets in the middle layer, and two α-helices in the lower layer, which resembles a Rossmann fold. Structural comparison suggested that AtTLP18.3 might be a phosphatase. The enzymatic activity of AtTLP18.3 was further confirmed by phosphatase assay with various substrates (e.g. p-nitrophenyl phosphate, 6,8-difluoro-4-methylumbelliferyl phosphate, O-phospho-L-serine, and several synthetic phosphopeptides). Furthermore, we obtained the structure of AtTLP18.3 in complex with O-phospho-L-serine to identify the binding site of AtTLP18.3. Our structural and biochemical studies revealed that AtTLP18.3 has the molecular function of a novel acid phosphatase in the thylakoid lumen. DUF477 is accordingly renamed the thylakoid acid phosphatase domain.
authors: Institute of Plant Biology, National Taiwan University, Taipei 106, Taiwan, Republic of China.
authors: Hsin-Yi
authors: HY
authors: Wu
authors: Mao-Sen
authors: MS
authors: Liu
authors: Tsan-Piao
authors: TP
authors: Lin
authors: Yi-Sheng
authors: YS
authors: Cheng
mesh_terms: D000135:Acid Phosphatase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002940:Circadian Rhythm / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D018360:Crystallography, X-Ray; D000458:Cyanobacteria / Q000201:enzymology; D057075:Enzyme Assays / Q000379:methods*; D018506:Gene Expression Regulation, Plant; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010768:Phosphoserine / Q000737:chemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D016415:Sequence Alignment; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D020524:Thylakoids / Q000201:enzymology*
chemical_list: D029681:Arabidopsis Proteins; D012333:RNA, Messenger; C523194:TLP18.3 protein, Arabidopsis; D010768:Phosphoserine; D000135:Acid Phosphatase