title: Control of catalysis in globin coupled adenylate cyclase by a globin-B domain.
abstract: The globin coupled heme containing adenylate cyclase from Leishmania major (HemAC-Lm) has two globin domains (globin-A and globin-B). Globin-B domain (210-360 amino acids) may guide the interaction between globin-A and adenylate cyclase domains for the regulation of catalysis. We investigated the role of globin-B domain in HemAC-Lm by constructing a series of mutants namely Δ209 (209 amino acids deleted), Δ360 (360 amino acids deleted), H161A, H311A and H311A-Δ209. Spectroscopic data suggest that the Δ209 and H311A-Δ209 proteins to be Fe(2+)-O2 form and apo form, respectively, indicating that His311 residue in the globin-B domain is crucial for heme binding in Δ209 protein. However, the H311A mutant is still of the Fe(2+)-O2 form whereas H161A mutant shows the apo form, indicating that only His161 residue in the globin-A domain is responsible for heme binding in full length enzyme. cAMP measurements suggest that the activities of Δ360 and Δ209 proteins were ∼10 and ∼1000 times lesser than full length enzyme, respectively, leading to the fact that globin-B domain inhibited catalysis rather than activation in absence of globin-A domain. These data suggest that the O2 bound globin-A domain in HemAC-Lm allows the best cooperation of the catalytic domain interactions to generate optimum cAMP.
authors.affiliation: Division of Structural Biology and Bio-informatics, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700 032, India.
authors.forename: Jayasree
authors.initials: J
authors.lastname: Roy
authors.affiliation: Division of Structural Biology and Bio-informatics, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700 032, India.
authors.forename: Sumit
authors.initials: S
authors.lastname: Sen Santara
authors.affiliation: Division of Structural Biology and Bio-informatics, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700 032, India.
authors.forename: Ayan
authors.initials: A
authors.lastname: Adhikari
authors.affiliation: Division of Structural Biology and Bio-informatics, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700 032, India.
authors.forename: Aditi
authors.initials: A
authors.lastname: Mukherjee
authors.affiliation: Division of Structural Biology and Bio-informatics, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700 032, India. Electronic address: adaks@iicb.res.in.
authors.forename: Subrata
authors.initials: S
authors.lastname: Adak
mesh_terms: D000262:Adenylyl Cyclases / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D004789:Enzyme Activation; D004926:Escherichia coli / Q000378:metabolism*; D005914:Globins / Q000737:chemistry* / Q000378:metabolism*; D006418:Heme / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D006418:Heme; D005914:Globins; D000262:Adenylyl Cyclases
keywords: Adenylate cyclase; Gas sensor proteins; Heme protein; Leishmania; Mutation; Spectroscopic studies