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    • Protein blast analysis of the

    2024-02-26

    Protein blast analysis of the sequence of globin-B with those of other Ketoconazole australia suggests that the globin-B domain in the HemAC-Lm displays only 6% identity and 34% similarity with that of cytoglobin/neuroglobin (Fig. 2). Furthermore, SWISS-MODEL protein modeling also predicts that the His311 is the proximal iron-coordinating ligand. The most significant feature is that the globin-B domain sequence lacks the Ketoconazole australia distal His residue, crucial residue for the endogenous distal axial ligand in the ferrous state of neuroglobin. Although the structure of the globin-B domain in the HemAC-Lm is similar to neuroglobin yet it lacks the entire D-helix. Based on the homology alignment data, we have speculated that the globin-B domain may have heme binding capacity. To investigate the role of the connecting globin-B domain, the globin-A domain deleted protein (Δ209) was constructed and compared with full length (HemAC-Lm) as well as catalytic domain (Δ360 HemAC-Lm) protein. In the present investigation, cloning, overexpression, and purification of the Δ209 HemAC-Lm protein were done and the characterization of its catalytic and physicochemical properties were studied in vitro as well as in vivo system.
    Materials and methods
    Results
    Discussion The primary sequence of the HemAC-Lm was found to be comprised of a connecting globin-B domain at the middle position between globin-A domain and catalytic AC domains. It has been established that the general role of connecting domain is to control the catalytic activity by the activation or auto inhibition of the enzyme [27]. The novel aspects those set the Δ209 apart from its full length enzyme as well as catalytic domain counterpart (Δ360) provide following information. (i) The Δ209 is catalytically inactive whereas the Δ360 has low activity (catalytically active) compared to full length enzyme; (ii) the oxygenated form of ferrous heme can nonspecifically bind to the globin-B domain of Δ209 protein, whereas the Δ360 is incapable of binding heme; (iii) in case of full length enzyme, the oxygenated form of heme can only bind to the globin-A domain but not to the globin-B domain; (iv) the ferrous state of Δ209 enzyme is five coordinate high spin whereas the full length enzyme shows six coordinate low spin state. The above properties of the connecting globin-B domain of this enzyme provide a unique perspective on HemAC-Lm structure-function. The Rz value (intensity ratios of absorption at 414 and 280nm) may be used to estimate the extent of heme binding to the protein (Table 1). If the Rz value is decreased upon mutation, heme binding affinity to the protein should be lowered. The Rz value of the H311A mutant in Δ209 enzyme is very negligible indicating that the His311 residue within the globin-B domain is the proximal axial ligand of heme in Δ209 enzyme. On the other hand, the Rz value of the H311A mutant in full length enzyme is found to be similar to that of wild type full length enzyme suggesting that the N-terminal globin-A domain prevents heme binding at His311 residue within globin-B domain. However, the O2 bound heme containing Δ209 protein and heme free H311A-Δ209 mutant protein displayed no AC activity. These results further prove that O2 bound heme binds nonspecifically to the globin-B domain in Δ209 protein. On the basis of catalytic activity, two types of regulations might have occurred by globin-A domain (1–209 aa) in HemAC-Lm. The ferric (wild type), ferrous (wild type), the heme-free form (H161A variant) of HemAC-Lm and catalytic domain (Δ360) proteins showed very low AC activity compared to oxygenated form of HemAC-Lm [5], [14] (Table 1) whereas Δ209 variants displayed no activity in vitro as well as in vivo condition. These results indicate that the globin-A domain (ferric, ferrous, the heme-free form of HemAC-Lm) relieves the connecting globin-B domain (209–360 aa) induced inhibition and oxygen binding to the ferrous state of the globin-A domain releases the suppression further within the catalytic domain (360–616 aa). Hence it appears another question that how the globin-A domain prevents the connecting globin-B domain induced inhibition. Basically the behavior of the connecting globin-B domain is similar to globin-A domain with respect to primary structure and O2 binding properties. We suspect that the globin-A domain interacts with catalytic domain at the interacting site of globin-B domain. Thus, the interaction of the globin-B domain with catalytic domain is not possible in presence of the globin-A domain of full length enzyme.