Expression and Characterization of HgcA and HgcB, Two Proteins Involved in Methylmercury Biosynthesis

methylmercury;biogeochemistry;mercury binding protein;environmental toxic element cycles;iron-sulfur protein;cobalamin (vitamin B12);Biological Chemistry;Science;Chemical Biology

Rush, KatherineSmith, Janet L ;

University of Michigan

关键词: methylmercury;    biogeochemistry;    mercury binding protein;    environmental toxic element cycles;    iron-sulfur protein;    cobalamin (vitamin B12);    Biological Chemistry;    Science;    Chemical Biology;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/147540/kwrush_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Methylmercury biosynthesis is a biologically-mediated process linked to expression of the recently discovered hgcAB gene products. The environmental conversion of toxic Hg(II) to MeHg, which exhibits even greater biological toxicity, is of concern to ecological food webs and piscivorous human populations. The mechanism by which HgcA and HgcB are responsible for Hg(II) methylation is unknown, but may represent a departure from canonical enzymatic methylation via carbocation mechanisms. The hgcAB genes are always found in genomic proximity to one another and predicted to interact within distances amenable to electron transfer in the proposed mechanism of Hg methylation. HgcA is a 40kDa protein comprised of cytosolic and transmembrane domains; the cytosolic domain is predicted to bind a cobalamin cofactor, based on its sequence homology to a known cobalamin-binding protein, which is suggested to serve as the catalytic center of the mercury methylation reaction. HgcB contains two [4Fe-4S] clusters and three additional conserved cysteine residues which we hypothesize are responsible for binding divalent mercury. No in vitro study of HgcA or HgcB has yet been conducted to our knowledge, and this dissertation presents the first protocols for the heterologous expression of both HgcA and HgcB. HgcA contains a C-terminal transmembrane domain and both proteins contain metal cofactors which present significant barriers to heterologous production; thus, Escherichia coli cell lines containing co-expression plasmids for cofactor assembly were constructed to circumvent these challenges. In Chapter 2, the HgcB protein was expressed and purified as a maltose-binding protein fusion with 2 iron-replete [4Fe-4S] clusters, as determined by UV-visible and EPR spectroscopic techniques. A series of HgcB variants were constructed containing alanine or serine substitution of the cysteine residues hypothesized to comprise a mercury binding site. The mercury binding characteristics of these variants were probed using stopped-flow rapid kinetics and the results indicated agreement with the requirement for cysteine 73 and either cysteine 94 or 95 observed in vivo. This study (a) indicates that these C-terminal cysteine residues are involved in the interaction of HgcB and mercury, and (b) establishes a production system to facilitate further exploration of this role. It was found in Chapter 3 that truncated HgcA constructs lacking the C-terminal binding domain are not competent to bind cobalamin cofactor. The full-length HgcA protein was expressed in the presence of high intracellular cobalamin and preliminary UV-visible characterization of the holo-FL-HgcA protein is presented. This expression and purification method comprises the critical first step toward in vitro validation of the functionality of HgcA as a mercury methylase, as well as the finding that the C-terminal transmembrane domain of HgcA is required for cofactor binding. These studies provide the first characterization of purified HgcA and HgcB, components of a biological system with the puzzling role of synthesizing highly toxic MeHg.