Mechanisms of chloroperoxidase-catalyzed enantioselective reactions as probed by site-directed mutagenesis and isotopic labeling

Chloroperoxidase (CPO) is a heme-containing glycoprotein secreted by the marine fungus Caldariomyces fumago. Chloroperoxidase contains one ferriprotoporphyrin IX prosthetic group per molecule and catalyzes a variety of reactions, such as halogenation, peroxidation and epoxidation. The versatile catalytic activities of CPO coupled with the increasing demands for chiral synthesis have attracted an escalating interest in understanding the mechanistic and structural properties of this enzyme. In order to better understand the mechanisms of CPO-catalyzed enantioselective reactions and to fine-tune the catalytic properties of chloroperoxidase, asparagine 74 (N74) located in the narrow substrate access channel of CPO was replaced by a bulky, nonpolar valine and a polar glutamine using site-directed mutagenesis. The CPO N74 mutants displayed significantly enhanced activity toward nonpolar substrates compared to wild-type CPO as a result of changes in space and polarity of the heme distal environment. More interestingly, N74 mutants showed dramatically decreased chlorination and catalase activity but significantly enhanced epoxidation activity as a consequence of improved kinetic perfection introduced by the mutation as reflected by the favorable changes in k cat and kcat/KM of these reactions. It is also noted that the N74V mutant is capable of decomposing cyanide, the most notorious poison for many hemoproteins, as judged by the unique binding behavior of N74V with potassium cyanide. Histidine 105 (H105) was replaced by a nonpolar amino acid alanine using site-directed mutagenesis. The CPO H105 mutant (H105A) displayed dramatically decreased chlorination and catalase activity possibly because of the decreased polarity in the heme distal environment and loss of the hydrogen bonds between histidine 105 and glutamic acid 183. However, significantly increased enantioselectivity was observed for the epoxidation of bulky styrene derivatives. Furthermore, my study provides strong evidence for the proposed histidine/cysteine ligand switch in chloroperoxidase, providing experimental support for the structure of the 420-nm absorption maximum for a number of carbon monoxide complexes of heme-thiolate proteins. For the NMR study, [dCPO(heme)] was produced using 90% deuterated growth medium with excess heme precursors and [dCPO(Phe)] was grown in the same highly deuterated medium that had been supplemented with excess natural phenylalanine. To make complete heme proton assignments, NMR spectroscopy has been performed for high-resolution structural characterization of [dCPO(heme)] and [dCPO(Phe)] to achieve unambiguous and complete heme proton assignments, which also allows important amino acids close to the heme active center to be determined.

Mechanisms of Arsenic Detoxification and Resistance

Arsenic is a ubiquitous environmental toxic substance. As a consequence of continual exposure to arsenic, nearly every organism, from Escherichia coli to humans have evolved arsenic detoxification pathways. One of the pathways is extrusion of arsenic from inside the cells, thereby conferring resistance. The R773 arsRDABC operon in E. coli encodes an ArsAB efflux pump that confers resistance to arsenite. ArsA is the catalytic subunit of the pump, while ArsB forms the oxyanion conducting pathway. ArsD is an arsenite metallochaperone that binds arsenite and transfers it to ArsA. The interaction of ArsA and ArsD allows for resistance to As(III) at environmental concentrations. The interaction between ArsA ATPase and ArsD metallochaperone was examined. A quadruple mutant in the arsD gene encoding a K2A/K37A/K62A/K104A ArsD is unable to interact with ArsA. An error-prone mutagenesis approach was used to generate random mutations in the arsA gene that restored interaction with the quadruple arsD mutant in yeast two-hybrid assays. Three such mutants encoding Q56R, F120I and D137V ArsA were able to restore interaction with the quadruple ArsD mutant. Structural models generated by in silico docking suggest that an electrostatic interface favors reversible interaction between ArsA and ArsD. Mutations in ArsA that propagate changes in hydrogen bonding and salt bridges to the ArsA-ArsD interface also affect their interactions. The second objective was to examine the mechanism of arsenite resistance through methylation and subsequent volatilization. Microbial ArsM (As(III) S-adenosylmethyltransferase) catalyzes the formation of trimethylarsine as the volatile end product. The net result is loss of arsenic from cells. The gene for CrArsM from the eukaryotic green alga Chlamydomonas reinhardtii was chemically synthesized and expressed in E. coli. The purified protein catalyzed the methylation of arsenite into methyl-, dimethyl- and trimethyl products. Synthetic purified CrArsM was crystallized in an unliganded form. Biochemical and biophysical studies conducted on CrArsM sheds new light on the pathways of biomethylation. While in microbes ArsM detoxifies arsenic, the human homolog, hAS3MT, converts inorganic arsenic into more toxic and carcinogenic forms. An understanding of the enzymatic mechanism of ArsM will be critical in deciphering its parallel roles in arsenic detoxification and carcinogenesis.