The mechanism study of fumitremorgin B dioxygenase and engineered human cystathionine gamma lyase

Date

2016-05

Authors

Yan, Wupeng

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Abstract

Peroxy-containing compounds represent a large class of natural products with many demonstrated beneficial effects to human health. Yet, very little is known about how endoperoxide functionality is incorporated into the natural products. In the first section of this dissertation, we have done the biochemical and structural research on the protein Fumitremorgin B dioxygenase, which is the first non-heme iron enzyme catalyzing an endoperoxide formation reaction. This work discloses mechanistic understanding to explain this unprecedented transformation. Distinct from all currently known α-ketoglurarate-dependent mononuclear non-heme iron enzymes, FtmOx1 incorporates molecular oxygen into the product without O-O bond scission, suggesting a novel mechanism. Indeed, the structural data reveal a surprising and unique arrangement of α-ketoglutarate (α-KG). Once the co-factor α-KG binds to the iron center, the remaining site for oxygen binding and activation is completely shielded from substrate access. This is in dramatic contrast to currently characterized α-ketoglurarate-dependent mononuclear non-heme iron enzymes, in which the oxygen binding site directly faces the substrate to be oxidized. From the crystal structure, we identify a tyrosine residue (Y224) as the residue shielding the mononuclear iron center from substrate access. The following biochemical study has shown that upon Y224A and Y224F mutation, the reaction is shifted from endoperoxide formation to a traditional α-ketoglurarate-dependent mononuclear non-heme iron enzyme catalyzed oxidative hydroxylation reaction. Further EPR study and pre-steady state analysis suggested an organic radical formed during catalysis. Those structural and biochemical data allow us to formulate a mechanistic model to account for this unprecedented endoperoxide formation reaction in which Y224 will form a tyrosyl radical and acting as a bridge to connect between the iron center and the substrate.

Cancer cells exhibit different metabolism compared to normal tissue, this has been shown to be a successful target in clinical. It has been found that some types of cancer are dependent on particular amino acids since they are not able to synthesize these amino acids themselves. Thus the strategy of starving tumor cell from its specific essential amino acid has great potential in anti-tumor therapeutic development. To consume the essential amino acid L-Methionine, human cystathionine-γ-lyase has been engineered to utilize methionine as substrate. One of the variants (hCGL-NLV) derived from this strategy showed altered specificity from cystathionine to methionine with improved half life compared with bacterial methionine gamma lyase. To understand the structural rationale to direct further bioengineering design, we obtained the crystal structures of this variant CGL-NLV in both an active and inactive conformations. The comparison between the two forms of hCGL-NLV highlighted a salt bridge between active site essential arginine residue (R62) and co-factor PLP that is attenuated upon high salt concentration. Mutation of this Arg to Alanine and Cysteine to eliminate salt bridge prevents the formation of the active configuration of CGL as well as its mutation variants which leads to the total loss of its enzymatic activity. Furthermore, the activity can be salvaged by chemical modification on protein residues to reinstall the electrostatic forces. Our results reveal CGL and all variants that exploit their ability to consume amino acids are highly sensitive to ion strength of the environment and emphasize it to be evaluated for bioengineering purpose in order to guarantee the optimal performance of the variants in vivo.

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