Date of Degree

2-2015

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Eleanore T. Wurtzel

Subject Categories

Biology | Molecular Biology

Abstract

Vitamin A deficiency is a widespread health issue in the tropics. To solve this issue, efforts are underway to increase provitamin A carotenoids such as β-carotene in staple crops which can be achieved by breeding, metabolic engineering or a combination of both approaches. However, rational strategies to improve carotenoid content in crops require sufficient knowledge of pathway regulation. Therefore, to better understand how plants synthesize provitamin A and to guide metabolic engineering strategies in crops such as maize, the functional characterization of the new ζ-carotene isomerase (Z-ISO) is of significant importance.

Z-ISO was recently discovered in maize and Arabidopsis (Chen et al., 2010). This new enzyme is a 15-cis-ζ-carotene isomerase present in all plants, diatoms and algae. Z-ISO is required in both green and non-green tissues including roots and the seed endosperm which is target for provitamin A biofortification. In this dissertation, to gain a better understanding of the role of Z-ISO in the isomerization of 15-cis-ζ-carotene, the Z-ISO polypeptide was biochemically characterized using extensive spectroscopy and its function was examined by developing an in vitro enzymatic assay and by an in vivo complementation system in Escherichia coli.

Bioinformatic tools modeled Z-ISO as an integral membrane and heme or non-heme iron binding protein. Therefore, using the Z-ISO polypeptide sequence, we selected conserved putative residue ligands for heme and non-heme iron and mutagenized them to Alanine. These Z-ISO mutant versions were tested for enzymatic function using an E. coli complementation system. These experiments showed that from all the conserved histidines present in Z-ISO, two (H150 and H266) as well as one aspartic acid residue (D294), were essential for isomerase activity. The only cysteine (C263) residue present in Z-ISO was not required for activity. These results are in good agreement with the predicted Z-ISO model where the locations for H150 and H266 are consistent with the coordination of a common factor.

Maize Z-ISO was also over-expressed and purified as a TEV protease-cleavable, maltose binding protein (MBP) fusion (MBP::Z-ISO). An in vitro assay utilizing substrate containing liposomes was developed to test Z-ISO activity. The conversion of the substrate 9,15,9'-tri-cis-ζ-carotene into the product 9,9'-di-cis-ζ-carotene by Z-ISO proceeded under reducing conditions but not under oxidizing conditions.

MBP::Z-ISO purified protein was also tested for the presence of metals. Using inductively coupled plasma optical emission spectrometry (ICP-OES) iron was detected but there were no significant levels of Ca, Cu, Mg, Mn, Mo, or Zn. Therefore, we concluded that Z-ISO is a metalloprotein. E. coli culture pellets expressing MBP::Z-ISO are brown consistent with the presence of iron or heme.

The presence of heme in purified MBP::Z-ISO was evaluated using heme staining and pyridine hemochrome assays. Heme staining of protein separated in SDS-PAGE gels revealed that MBP::Z-ISO and Z-ISO contain heme while MBP alone does not. Moreover, hemochrome assays showed the presence of heme b in Z-ISO. The UV-visible absorption spectrum of the intact, as isolated Z-ISO confirmed the presence of heme b in the oxidized, ferric Fe (III) state. Also, the spectrum of reduced Z-ISO is similar to that of cytochromes containing heme b with two axial histidine ligands and the reduced heme b binds carbon monoxide (CO).

Z-ISO was also characterized using electron paramagnetic resonance (EPR). An X-band spectrum of the MBP::Z-ISO fusion detected high-spin ferric heme (e.g. heme b with a single histidine ligand), multiple low-spin heme species, and a non-heme iron center. EPR also indicated the presence of two low-spin heme species and that one of these hemes might have a bis-histidine coordination and the other might have a histidine-cysteine axial coordination. Reduced Z-ISO also binds NO which is consistent with the binding of CO.

To gain more details on the heme cofactor, the same sample used in the EPR experiments was used to characterize the heme iron in MBP::Z-ISO using magnetic circular dichroism (MCD) which detects only heme iron. MCD characterization showed that Z-ISO has two ligand pairs (His/His and His/Cys), a result that is consistent with the EPR results. MCD experiments also showed a redox-dependent change in ligand (Cys↔His) coordination of a low-spin heme b. Our data also suggested the existence of a high-spin, 5-coordinate, His-ligated heme which was detected by EPR and as a minor species by MCD. We hypothesized that substrate could bind to this intermediate or that substrate displaces an axial ligand to coordinate with the heme iron. MCD, EPR, and UV-Vis analysis showed that exogenous ligands bind the Fe(II) state but it was not known whether an exogenous ligand can displace an axial ligand when the heme is in the Fe(III) state. To test this possibility we utilized addition of cyanide (CN-), which binds preferably to the ferric rather than the ferrous heme. CN- was added to both the as isolated (oxidized) Fe(III) ferric enzyme and to the Fe(II) (dithionite-reduced) ferrous enzyme. UV-Vis absorption spectra indicated CN- binding to the ferric Fe(III) heme which suggest that Z-ISO can also bind exogenous ligands in the oxidized form. Taken together our data suggest that heme is essential for Z-ISO activity. The presence of heme and its requirement for Z-ISO activity are surprising since heme is not often described in isomerization reactions. Based on our results, we propose a mechanism for Z-ISO function.

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