A Novel Pathway for the Biosynthesis of Heme in Archaea: Genome-Based Bioinformatic Predictions and Experimental Evidence
Figure 4
Purification and characterization of Mba_1791 and Mba_1461. (a) SDS-PAGE analysis of purified Mba_1791 (lane 1) and Mba_1461 (lane 2). Gel permeation chromatography revealed native relative molecular masses of 55,300 ± 840 Da for Mba_1791 (dotted line) and 60,800 ± 7,300 Da for Mba_1461 (solid line), respectively. (b) UV-visible absorption spectrum of extracted tetrapyrroles which accumulated during production of recombinant Mba_1791 in E. coli. (c) UV-visible absorption spectra of enzyme assays after overnight incubation at 37°C in the anaerobic chamber. Uroporphyrinogen III was produced from ALA by the enzymes HemB, HemC, and HemD (dashed double dotted line). Addition of purified Mba_1791 and SAM to the assay mixture resulted in precorrin-2 formation (solid line). Addition of purified NirE, Mba_1461, and NAD+ to the assay resulted in formation of sirohydrochlorin (dotted line). In a coupled enzyme assay containing purified Mba_1791 and Mba_1461 the formation of sirohydrochlorin was also observed (dashed line). For exact details see Section 2.
