title: In silico analysis and experimental validation of lipoprotein and novel Tat signal peptides processing in Anabaena sp. PCC7120.
abstract: Signal peptide (SP) plays a pivotal role in protein translocation. Lipoprotein- and twin arginine translocase (Tat) dependent signal peptides were studied in All3087, a homolog of competence protein of Synechocystis PCC6803 and in two putative alkaline phosphatases (ALPs, Alr2234 and Alr4976), respectively. In silico analysis of All3087 is shown to possess the characteristics feature of competence proteins such as helix-hairpin-helix, N and C-terminal HKD endonuclease domain, calcium binding domain and N-terminal lipoprotein signal peptide. The SP recognition-cleavage site in All3087 was predicted (AIA-AC) using SignalP while further in-depth analysis using Pred-Lipo and WebLogo analysis for consensus sequence showed it as IAA-C. Activities of putative ALPs were confirmed by heterologous overexpression, activity assessment and zymogram analysis. ALP activity in Anabaena remains cell bound in log-phase, but during late log/stationary phase, an enhanced ALP activity was detected in extracellular milieu. The enhancement of ALP activity during stationary phase was not only due to inorganic phosphate limitation but also contributed by the presence of novel bipartite Tat-SP. The Tat signal transported the folded active ALPs to the membrane, followed by anchoring into the membrane and successive cleavage enabling transportation of the ALPs to the extracellular milieu, because of bipartite architecture and processing of transit Tat-SP.
authors.affiliation: Molecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai, 400085, India.
authors.forename: Sonika
authors.initials: S
authors.lastname: Kumari
authors.affiliation: Molecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai, 400085, India. chaurasia@skku.edu.
authors.forename: Akhilesh Kumar
authors.initials: AK
authors.lastname: Chaurasia
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D017033:Anabaena / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D003198:Computer Simulation; D055029:Inverted Repeat Sequences; D008074:Lipoproteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010710:Phosphates / Q000378:metabolism; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D015398:Signal Transduction; D000069294:Twin-Arginine-Translocation System / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D008074:Lipoproteins; D010710:Phosphates; D021382:Protein Sorting Signals; D000069294:Twin-Arginine-Translocation System; D000469:Alkaline Phosphatase
keywords: Anabaena sp. PCC7120; Sec pathway; bipartite signal peptide; lipoprotein signal peptide; protein transport; twin arginines translocase