Abstract A data base search using the amino acid sequence of Saccharomyces cerevisiae Etr1p, the last enzyme of mitochondrial fatty acid synthesis type II (FAS II), revealed a highly similar human protein, NRBF-1. Expression of NRBF-1 in a yeast etr1Δ strain rescued its respiratory deficiency. NRBF-1 resides in mitochondria in cultured HeLa cells. The recombinant NRBF-1 is enzymatically active, reducing 2E-enoyl-CoAs to acyl-CoAs in an NADPH-dependent manner. Altogether, our data showed that NRBF-1 is a mitochondrial 2-enoyl-CoA reductase/2-enoyl thioester reductase (MECR/ETR1), the human functional counterpart of yeast Etr1p. In addition, MECR was also isolated from bovine heart. It turns out that mammals contain a mitochondrial FAS II pathway, in addition to cytoplasmic FAS I. To investigate the functional mechanism of MECR/ETR1 at the molecular level, the protein was crystallized and the crystal structure determined. The apo-structure of MECR/ETR1 contains two sulfates in the nucleotide binding site and the domain arrangement resembles the NADPH-containing holo-structure of yeast Etr1p. The predicted mode of NADPH-binding and kinetic data suggest that Tyr94 and Trp311 play critical roles in catalysis. A pocket was found in the structure extending away from the catalytic site that can accommodate fatty acyl chains up to 16 carbons. An acyl carrier protein (ACP) binding site was also suggested. To study the physiological function of mouse Mecr, two lines of transgenic mice overexpressing Mecr were generated. The Mecr transgenic mice developed cardiac and mitochondrial abnormalities. The phenotyping was carried out using echocardiography, heart perfusion, histology, and endurance testing. Our results suggest Mecr plays a role in mitochondrial and heart function. Therefore, inappropriate expression of the genes of FAS II may result in the development of cardiomyopathy.

The exoglucanase from Cellulomonas fimi catalyses the hydrolysis of cellobiose units from the non-reducing terminus of cello-oligosaccharides with overall retention of anomeric configuration. Its mechanism of action is therefore thought to involve a double displacement reaction, involving as the first step, formation of a glycosyl-enzyme intermediate (glycosylation) and as a second step, the hydrolysis of this intermediate (deglycosylation). This mechanism is investigated here through the study of the kinetics of hydrolysis of aryl β-glucosides and aryl β-cellobiosides and by employing the mechanism-based irreversible inactivators, 2', 4'-dinitrophenyl 2-deoxy-2-fluoro-β-D-glucoside (2F-DNPG) and 2", 4"-dinitrophenyl 2-deoxy-2-fluoro-β-D-cellobioside (2F-DNPC). The study with the aryl β-glucosides revealed that this enzyme is indeed active on glucosides, a feature that had previously been undetected. A linear relationship was found to exist between the logarithm of Vmax for hydrolysis and the phenol pKa as well as between the logarithm of Vmax/Krn and me phenol pKa, showing that glycosylation is both the rate determining step and the first irreversible step for all substrates. The reaction constant calculated, ρ = 2.21, indicates a considerable amount of charge build up at the transition state of glycosylation. The linear free energy relationship study of the aryl β-cellobiosides revealed no significant dependence of the logarithm of Vmax on the pKa of the phenol, indicating that deglycosylation is rate determining. However, the slight downward trend in this Hammett plot at higher pKa values may suggest that the rate determining step is changing from deglycosylation to glycosylation. However, the logarithm of Vmax/Km does correlate with the pKa of the phenol, thus showing that the first irreversible step is glycosylation. The reaction constant (ρ = 0.60) which reflects the development of charge at the glycosylation transition state for the cellobiosides is less than that calculated for the glucosides, thus suggesting a glycosylation transition state with either a greater degree of acid catalysis or less C-O bond cleavage than that for the glucosides. The inactivators, 2F-DNPC and 2F-DNPG, are believed to inactivate the exoglucanase by binding to the enzyme and forming covalent glycosyl-enzyme intermediates. The inactivated-enzyme was stable in buffer but reactivated in the presence of a suitable glycosyl-acceptor such as cellobiose, presumably via a transglycosylation reaction. These results indicate that covalent 2F-glycosyl-exoglucanase intermediates are stable and are catalytically competent to turn over to product, thus supplying further evidence for the Koshland mechanism. The exoglucanase is inactivated more rapidly by 2F-DNPC than by 2F-DNPG. However, both inactivated forms of the enzyme reactivated at comparable rates in the presence of cellobiose, showing that the second glucosyl unit present on the cellobiosides increases the rate of glycosylation relative to that found for the glucosides but not the rate of deglycosylation. The stable covalent nature of the 2F-glycosyl-enzyme intermediates provided an excellent opportunity to identify the enzymic nucleophile. This was accomplished by radiolabelling the exoglucanase with a tritiated analogue of 2F-DNPG cleaving the protein into peptides and purifying the radiolabelled peptides. Sequencing of this peptide resulted in the identification of the active site nucleophile as glutamic acid residue 274. This residue was found to be highly conserved in this family of β-glycanases, further indicating its importance in catalysis. / Science, Faculty of / Chemistry, Department of / Graduate

Recent reports in the literature suggest that oral contraceptive steroid therapy may be implicated in the development of benign hepatic adenomas in women. Since estrogens and progestins are known to affect liver function, we studied effects of chronic administration of the oral contraceptive agents mestranol and norethindrone on various indices of hepatic integrity. Several hepatic mixed function oxidase activities were measured: benzo(a)pyrene hydroxylase, epoxide hydrase, aniline hydroxylase (Appendix II) and aminopyrlne N-demethylase (Appendix II). In addition, benzo(a)pyrene hydroxylase activity in lung tissue was measured. As an indication of whether metabolites of the contraceptive steroids were bound to liver macromolecules, irreversible binding of [³H]-benzopyrene was measured. Hepatic histopathology (light microscopy using hematoxylin and eosin stain and oil red 0 stain) was carried out to determine if functional alterations correlated with pathological changes in the liver. Comparisons were also made between ethanol treated and non-ethanol treated groups to determine if contraceptive steroid-associated hepatotoxiclty was enhanced by or would enhance, the hepatotoxiclty of ethanol administration. Female and male Wistar rats were pair-fed a nutritional liquid diet, Sustacal[sup R] (Mead Johnson) to which was added either sucrose or ethanol as 40% of calories. Oral contraceptive steroids were administered daily in the liquid, diet in the following doses: mestranol, 0.6 mg/kg per day, alone or in combination with norethindrone, 5.0 mg/kg per day. Initial short-term studies showed that the ethanol plus Sustacal[sup R] diet generally caused enzyme induction compared to the plain Sustacal[sup R] diet or the sucrose plus Sustacal[sup R] diet in animals treated for up to 6 weeks. Animals that were administered the contraceptive steroids for a similar time period also demonstrated hepatic microsomal enzyme induction. Enzyme activity in animals that received ethanol plus the contraceptive steroids was increased above that seen for each agent alone. Chronic studies showed that ethanol administration for 6 months produced hepatotoxiclty in both male and female rats. Hepatotoxiclty was observed functionally as decreased hepatic benzo(a)pyrene hydroxylase activity and histopathologically as increased fat accumulation in zone 3 of the liver lobules. It was found that administration of the contraceptive steroids to female rats tended to protect against ethanol-associated hepatotoxiclty. The protective effect was observed functionally as maintenance of control levels of hepatic benzo(a)pyrene hydroxylase activity and morphologically as lesser amounts of fat accumulation ln the liver. That is, there tended to be a correlation between the level of hepatic benzo(a)pyrene hydroxylase activity and histological fat accumulation as an indication of ethanol-associated hepatotoxiclty. A Sustacal associated phenomenon was evident in all animals in which hlstopathology was carried out. The "Sustacal effect" was observed, as mlcrodroplet fat accumulation ln zone 1 of the liver lobule. Contraceptive steroid treated females showed the least "Sustacal effect". Microsomal enzyme activity did not appear to be affected by the "Sustacal effect". It was concluded that the contraceptive steroids administered did not increase ethanol hepatotoxicity. Instead, it appeared that female sex steroids tended to attenuate ethanol-assoclated hepatotoxicity. There was no evidence to suggest that the oral contraceptive steroids were directly associated with overt hepatotoxicity. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

Mononuclear molybdenum enzymes catalyze a variety of reactions that are essential in the cycling of nitrogen, carbon, arsenic, and sulfur. For decades, the structure and function of these crucial enzymes have been investigated to develop a fundamental knowledge for this vast family of enzymes and the chemistries they catalyze. The dimethyl sulfoxide reductase (DMSOR) family is the most diverse family of molybdoenzymes and, the members of this family catalyze a myriad of reactions that are important in microbial life processes. Periplasmic nitrate reductase (Nap) is an important member of the DMSO reductase family that catalyzes the reduction of nitrate to nitrite, and yet the physiological role of Nap is not completely clear. Enzymes in this family can transform multiple substrates; however, quantitative information about the substrate preference is sparse and more importantly, the reasons for the substrate selectivity are not clear. Substrate specificity is proposed to be tuned by the ligands coordinating the molybdenum atom in the active site. As such, periplasmic nitrate reductase is utilized as a vehicle to understand the substrate preference and delineate the mechanistic underpinning of these differences. To this end, NapA from <i>Campylobacter jejuni </i>has been heterologously overexpressed, and a series of variants, where the molybdenum-coordinating cysteine has been replaced with another amino acid, has been produced. The kinetic and biochemical properties of these variants will be discussed and compared with those of the native enzyme, providing quantitative information to understand the function.

No description available.

Nucleases are integral to all DNA processing pathways. The exact nature of substrate recognition and enzymatic specificity in structure-specific nucleases that are involved in DNA replication, repair and recombination has been under intensive debate. The nucleases that rely on the contours of their substrates, such as 5’ nucleases, hold a distinctive place in this debate. How this seemingly blind recognition takes place with immense discrimination is a thought-provoking question. Pertinent to this question is the observation that even minor variations in the substrate provoke extreme catalytic variance. Increasing structural evidence from 5’ nucleases and other structure-specific nuclease families suggest a common theme of substrate recognition involving distortion of the substrate to orient it for catalysis and protein ordering to assemble active sites. Using three single-molecule (sm)FRET approaches of temporal resolution from milliseconds to sub-milliseconds, along with various supporting techniques, I decoded a highly sophisticated mechanism that show how DNA bending and protein ordering control the catalytic selectivity in the prototypic system human Flap Endonuclease 1 (FEN1). Our results are consistent with a mutual induced-fit mechanism, with the protein bending the DNA and the DNA inducing a protein-conformational change, as opposed to functional or conformational selection mechanism. Furthermore, we show that FEN1 incision on the cognate substrate occurs with high efficiency and without missed opportunity. However, when FEN1 encounters substrates that vary in their physical attributes to the cognate substrate, cleavage happens after multiple trials During the course of my work on FEN1, I found a novel photophysical phenomena of protein-induced fluorescence quenching (PIFQ) of cyanine dyes, which is the opposite phenomenon of the well-known protein-induced fluorescence enhancement (PIFE). Our observation and characterization of PIFQ led us to further investigate the general mechanism of fluorescence modulation and how the initial fluorescence state of the DNA-dye complex plays a fundamental role in setting up the stage for the subsequent modulation by protein binding. Within this paradigm, we propose that enhancement and quenching of fluorescence upon protein binding are simply two different faces of the same process. Our observations and correlations eliminate the current inconvenient arbitrary nature of fluorescence modulation experimental design.

Indiana University-Purdue University Indianapolis (IUPUI) / Mononuclear molybdenum enzymes catalyze a variety of reactions that are essential in the cycling of nitrogen, carbon, arsenic, and sulfur. For decades, the structure and function of these crucial enzymes have been investigated to develop a fundamental knowledge for this vast family of enzymes and the chemistries they catalyze. The dimethyl sulfoxide reductase (DMSOR) family is the most diverse family of molybdoenzymes and, the members of this family catalyze a myriad of reactions that are important in microbial life processes. Periplasmic nitrate reductase (Nap) is an important member of the DMSO reductase family that catalyzes the reduction of nitrate (NO3-) to nitrite (NO2-), and yet the physiological role of Nap is not completely clear. Enzymes in this family can transform multiple substrates; however, quantitative information about the substrate preference is sparse and more importantly, the reasons for the substrate selectivity are not clear. Substrate specificity is proposed to be tuned by the ligands coordinating the molybdenum atom in the active site. As such, periplasmic nitrate reductase is utilized as a vehicle to understand the substrate preference and delineate the mechanistic underpinning of these differences. To this end, NapA from Campylobacter jejuni has been heterologously overexpressed, and a series of variants, where the molybdenum-coordinating cysteine has been replaced with another amino acid, has been produced. The kinetic and biochemical properties of these variants will be discussed and compared with those of the native enzyme, providing quantitative information to understand the function.

No description available.

Tobacco-soil bacteria have evolved not only to tolerate high concentrations of nicotine, but to degrade it as a primary growth source. The genomes of several of these species have been sequenced, allowing for the identification of unique bacterial degradation pathways. In the Gram-negative bacteria, Pseudomonas putida, the nicotine-degrading gene cluster has been described; the encoded enzymes catabolize nicotine via the pyrrolidine pathway, ultimately forming malate and fumarate. In previous studies, the flavoenzyme, nicotine oxidoreductase (NicA2), has been identified as the first committed step of nicotine catabolism in this organism. Preliminary kinetic analysis reported that NicA2 has high specificity for S-nicotine, but a slow catalytic rate. Taking advantage of its unique evolutionary adaptation, we aim to refine the inherent catalytic function and structural features of NicA2 towards the development of a biotherapeutic for nicotine addiction, nicotine poisoning and tools for nicotine biosensor development. Our goal is to identify the factors contributing to the mechanistic and substrate-binding properties of NicA2 to improve its biotherapeutic potential. This work presents the first crystal structure of NicA2, resolved to 2.2 Å resolution, establishing it as a member of the flavin-dependent amine oxidase family with a conserved amine oxidase fold. Structural analysis identified a unique composition of the canonical aromatic cage (W427 and N462), which flanks the flavin isoalloxazine ring. Additionally, the X-ray crystallographic structure of the NicA2/S-nicotine complex was refined to 2.6 Å resolution, revealing a hydrophobic active site in support of a hydride-transfer mechanism. Analysis of enzyme activity with a series of substrate analogs and kinetic analysis of active-site residues reveal the determinants of substrate binding affording the remarkable specificity of this enzyme. Using site-directed mutagenesis of aromatic cage residues, along with analysis of the kinetics of the reductive and oxidative steps, we demonstrate that the rate-limiting reaction step is in the oxidative half-reaction. Structural analysis of an active-site variant revealed a secondary binding site consistent with kinetic analysis demonstrating substrate inhibition. Together, our findings provide kinetic and structural evidence for the catalytic mechanism of NicA2, expanding the possibilities for the generation of catalytically-efficient variants and supporting its role as a promising therapeutic strategy. / 2021-01-30T00:00:00Z

<p dir="ltr">The Cdc14 phosphatase family is highly conserved in fungi. In Saccharomyces cerevisiae, Cdc14 is essential for down-regulation of cyclin-dependent kinase activity at mitotic exit. However, this essential function is not broadly conserved and requires only a small fraction of normal Cdc14 activity. In general, few conserved functions of Cdc14 phosphatase have been defined. Here, I present mechanistic biochemical and phenotypic characterization of Cdc14 phosphatases in fungi. I have demonstrated that fungal Cdc14 phosphatases possess an invariant motif in the disordered C-terminal tail that is required for full enzymatic activity. This motif, termed substrate-like catalytic enhancer (SLiCE), functions during the rate-limiting step of Cdc14-directed catalysis, by binding to the active site and supporting phospho-enzyme hydrolysis. Adjacent to the SLiCE motif exists a conserved minimal Cdk consensus motif that likely serves a regulatory function as phosphorylation of this site inhibits Cdc14 activity in vitro. Vertebrate Cdc14 enzymes also possess a distinct, but mechanistically similar SLiCE motif, which may be the first described biochemical difference between Cdc14 enzymes. Moreover, the vertebrate SLiCE motif lacks an adjacent Cdk consensus motif, which may point to differences in how Cdc14 activity is regulated in higher eukaryotes.</p><p dir="ltr">Mutation of this motif in vivo served as a tool to discover biological processes that require high Cdc14 activity. In S. cerevisiae strains expressing this hypomorphic mutant allele (cdc14hm), I discovered a novel sensitivity to cell wall stresses, including chitin-binding compounds and echinocandin antifungal drugs. This sensitivity was also observed in the distantly related fungi Schizosaccharomyces pombe deletion strain and the human fungal pathogen Candida albicans hypomorphic and deletion strains, suggesting that this phenotype reflects a conserved function of Cdc14 orthologs in mediating fungal cell wall integrity. I also revealed that high Cdc14 activity is required for C. albicans ability to develop hyphae, which is an important virulence trait. This led to our determination that high Cdc14 activity is critical for virulence in two animal models of invasive candidiasis. Together, these results argue that Cdc14 would be an excellent antifungal drug target for the treatment of invasive Candida infections and sensitization to existing antifungal drugs.</p><p dir="ltr">Lastly, I implemented the auxin-inducible degradation system in C. albicans. Using this system, we were able to deplete Cdc14 and other target protein levels to >95% within minutes. Depletion of Cdc14 was robust enough to phenocopy gene deletions, confirming previous results and demonstrating the utility of rapid target protein inactivation. This system will serve as a powerful tool for future functional characterization of Cdc14 in C. albicans and other pathogenic fungal species.</p>