Theses and Dissertations in Biochemistry Copyright (c) 2013 University of Nebraska - Lincoln All rights reserved. http://digitalcommons.unl.edu/biochemdiss Recent documents in Theses and Dissertations in Biochemistry en-us Thu, 25 Apr 2013 01:41:35 PDT 3600 Investigations of Substrate Channeling in the Proline Oxidative Pathway http://digitalcommons.unl.edu/biochemdiss/12 http://digitalcommons.unl.edu/biochemdiss/12 Tue, 23 Apr 2013 09:05:21 PDT In cell metabolism, substrate channeling is a phenomenon where the product of one reaction is transported to a second enzyme active site without equilibrating into bulk solvent. Chapter 1 reviews the rationale and evidence for substrate channeling with the specific example of proline metabolism. Oxidation of proline to glutamate is catalyzed in consecutive reactions by proline dehydrogenase (PRODH) and pyrroline-5-carboxylate dehydrogenase (P5CDH). The intermediate Δ1-pyrroline-5-carboxylate reportedly tends to be labile and inhibitory towards several metabolic pathways.

One of the main objectives of this dissertation was to investigate substrate channeling between independent proline oxidative enzymes from Thermus thermophilus- TtPRODH and TtP5CDH. Chapter 2 establishes that TtPRODH and TtP5CDH are capable of interacting with a dissociation constant (KD) of 3.03 µM as demonstrated using Surface Plasmon Resonance (SPR). As observed in the present study, this interaction is possible only with a specific orientation of TtPRODH relative to TtP5CDH. A docking model of the two enzymes predicts an orientation of the active sites which is supportive of substrate channeling. Corroborating observations are made with kinetic studies. We observe that interference of TtPRODH-TtP5CDH complex by catalytically inactive mutants TtPRODH R288M/R289M and TtP5CDH C322A lead to significant decrease in glutamate formation. The results pave the way for testing substrate channeling in eukaryotic enzymes. In chapter 3, two novel eukaryotic enzymes from Saccharomyces cerevisiae, Put1p (PRODH) and Put2p (P5CDH), have been characterized. Particular attention was focused on the oxidative half-reaction of Put1p for gaining insight into possible redox functions of human PRODH.

Previous studies show that bifunctional enzyme from Gram-negative Bradyrhizobium japonicum (BjPutA) containing PRODH and P5CDH domains, exhibits substrate channeling via an elegant internal tunnel. BjPutA and its channeling variants were used to test the role of substrate channel in hydrolysis of P5C, an essential step in proline oxidation. These aspects of substrate channeling are discussed in chapter 4.

Overall, this study provides an improved understanding of: (1) Substrate channeling in proline oxidation; and (2) a model for investigating substrate channeling between other individual enzymes that catalyze consecutive reactions.

Advisor: Donald F. Becker

]]> Nikhilesh Sanyal Phylogenetic Engineering of the Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Large Subunit in <i>Chlamydomonas Reinhardtii</i> http://digitalcommons.unl.edu/biochemdiss/11 http://digitalcommons.unl.edu/biochemdiss/11 Mon, 03 Dec 2012 12:04:02 PST Thirty-four residues in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) may account for the kinetic differences between Rubisco enzyme from green algae and land plants. By substituting these "phylogenetic residues" as groups and combinations of groups in the large subunit of the green alga Chlamydomonas reinhardtii with those of land-plant Rubisco, the functions and relationships of these "phylogenetic groups" were determined.

A phylogenetic-group substitution at the base of catalytic loop 6 of the large subunit decreases the CO2/O2 specificity of the enzyme, but function is restored by a further phylogenetic-group substitution at the carboxy-terminal tail. Therefore, these two regions of the large subunit, which sandwich loop 6, are complementary. In addition, combining substitutions at the base of loop 6 and the large/small-subunit interface region produces a mutant enzyme that has to be complemented by the land-plant small subunit for function in Chlamydomonas. On the other hand, substitutions in a-helix G of the large subunit reduce the holoenzyme level, and, because Chlamydomonas mutants with additional substitutions in α-helices 7 and 8 cannot be recovered as photosynthetic-transformants, the three α helices appear to influence holoenzyme assembly.

A previous study showed that substituting five large-subunit residues and a small-subunit loop with land-plant identities produced an enzyme (termed "penta/ABSO") with land-plant catalytic properties. In the present study, through structural dissection, it is concluded that all the residues substituted in penta/ABSO are required for the shift towards land-plant catalysis. Among the residues substituted in penta/ABSO is methyl-Cys-256, which indicates that posttranslational modifications of the large subunit may also play a role in catalysis. Further study of cysteine methylation and proline hydroxylation showed that mutations of methyl-Cys-256 and hydroxy-Pro-104 influence catalysis.

The current study complements previous knowledge about Rubisco, and provides further structural targets for the beneficial engineering of Rubisco.

Advisor: Robert J. Spreitzer

]]> Boon Hoe Lim Sinusoidal Endothelial Dysfunction in Non-Alcoholic Fatty Liver Disease. http://digitalcommons.unl.edu/biochemdiss/10 http://digitalcommons.unl.edu/biochemdiss/10 Mon, 03 Dec 2012 11:17:14 PST Non-alcoholic fatty liver disease (NAFLD) is an asymptomatic increasingly common disorder that affects liver metabolism and is often the precursor for liver pathologies such as fibrosis, cirrhosis and hepato-cellular carcinoma. The liver sinusoidal endothelial cells act as a liver sieve by allowing macromolecules and chylomicrons to traverse through their fenestrations (sieve plates) to hepatocytes. Since liver sinusoidal endothelial cells (LSEC) regulate serum derived macromolecular exposure to hepatocytes, we asked what role LSEC could play in the pathogenesis of NAFLD. To investigate the early events of NAFLD we used a rat model (Sprague-Dawley) in which animals were maintained on standard and high fat diets (HFD) for a period of 8 weeks. The lipid accumulation in the livers, isolated hepatocytes and LSEC were visualized by Oil Red O, BODIPY (boron-dipyrromethene) stains and CARS (Coherent Anti-stokes Raman Scattering) imaging. There was evidence of increased size of lipid droplets in the hepatocytes of HFD rats in contrast to their LSEC, which showed minimal to no lipid accumulation. The lipid content of the liver tissues was analyzed by thin layer chromatography, which revealed the accumulation of triglycerides and cholesteryl esters in the livers of HFD rats compared to the control rats. In vitro endocytosis of 125I-HA (hyaluronic acid) experiments carried out on LSECs isolated from the rat livers showed that the 125I-HA uptake is significantly higher in control rats compared to HFD rats. We also observed that serum HA levels and alkaline phosphatase (ALP) levels were increased in HFD rats, in contrast to alanine transaminase (ALT), triglycerides and cholesterol, which remained the same. Purified RNA from the isolated LSECs was subjected to microarray analysis demonstrating altered gene expression patterns between rats on two different diets. The kidneys, gut, and spleens were also harvested to study the interplay of the organs in the disease. We have been able to demonstrate that decreased endocytic ability of LSECs precedes fibrosis of liver in NAFLD.

Adviser: Edward N. Harris

]]> Sandhya Lakshmi Gopalakrishnan Developing a High Throughput Protocol for Using Soil Molecular Biology as Trace Evidence http://digitalcommons.unl.edu/biochemdiss/9 http://digitalcommons.unl.edu/biochemdiss/9 Tue, 01 May 2012 09:24:33 PDT The use of soil as trace evidence has changed significantly with the addition of new techniques. These techniques include using the biochemical molecules from soil microbial communities to make a fingerprint of the specific soil. This research examines the changes to the microbial community profile that take place during storage of a soil sample. To observe such changes both the DNA and fatty acid profiles will be examined.

The DNA profiles were made with capillary electrophoresis-single stranded conformation polymorphism (CE-SSCP). After statistical analysis using Bray-Curtis distances and ANOSIM (analysis of similarity) it was shown that storage of soil does not have a significant impact on the microbial community profile. However, when samples were compared across soil collection sites significant differences were seen. This illustrates that different soils respond differently to storage treatments.

The fatty acid profiles were analyzed as fatty acid methyl esters (FAMEs) using gas chromatography. Data were analyzed using canonical correlation analysis, squared Mahalanobis distance, and repeated measures. The results show that -80˚C is the best way to store soils to preserve the integrity of the microbial community FAME profile, followed by -20˚C. It was also demonstrated that when using fatty acids to examine the change within the soil at the collection site there is generally not a significant difference between the soil collected over a two week period.

When the two methods are compared FAME is a more sensitive method to minute changes within the microbial community. With the data from these two methods, using soil microbial community profiling is closer to becoming a viable option for forensic science.

Adviser: Cheryl P. Bailey

]]> Sabreena A. Larson Studies on the Small Ubiquitin-Like Modifier (SUMO) E2 Conjugases of the SUMOylation System in <i>Chlamydomonas reinhardtii</i> and their Role in Stress Physiology http://digitalcommons.unl.edu/biochemdiss/8 http://digitalcommons.unl.edu/biochemdiss/8 Tue, 24 Apr 2012 07:18:57 PDT The eukaryotic protein post-translational modification by SUMOylation is involved in a diverse array of cellular processes, including various stress responses. A fully functional SUMOylation system is present in the unicellular green alga Chlamydomonas reinhardtii, and SUMOylation of multiple high molecular weight proteins is induced in response to abiotic stress in this organism. We report here the characterization of a SUMO E2 conjugase deletion mutant in C. reinhardtii, mut5. SUMO E2 conjugase enzymes are responsible for the conjugation of the protein SUMO to a lysine residue within a target protein. C. reinhardtii mutants in which the SUMO E2 conjugase CrUBC9 has been deleted (mut5) fail to modify proteins with SUMO in response to multiple stress conditions, and this failure to SUMOylate generally results in a reduced tolerance to a given stress. Complementation of CrUBC9 mutants with the deleted gene demonstrates that CrUBC9 is solely responsible for SUMOylation under stress conditions, and that it predominantly localizes to the nucleus.

In addition, we identify the likely presence of two distinct SUMO E2 conjugase proteins in the C. reinhardtii genome. This is in marked contrast to virtually all other organisms studied to date, in which a single essential SUMO E2 conjugase has been identified. Bioinformatic analyses allowed the identification of the likeliest candidate for this second SUMO E2 conjugase, CrUBC3. Comparison of CrUBC3 and CrUBC9 reveals that they have distinct sequence features that distinguish them from other known SUMO E2 conjugases. In addition, we show that the transcripts encoding these proteins are regulated in a distinct and opposite manner in response to stress, consistent with separate functions for these proteins within the cell. Expression of these proteins in a heterologous yeast system allowed the examination of the functionality of these two proteins as SUMO E2 conjugases, and suggests that the two likely function in distinct, non-overlapping capacities within C. reinhardtii.

Advisor: Donald P. Weeks

]]> Amy R. Knobbe The crosstalk between human fatty acid transport protein 1 and fatty acid transport protein 4 http://digitalcommons.unl.edu/biochemdiss/7 http://digitalcommons.unl.edu/biochemdiss/7 Fri, 14 Oct 2011 06:37:31 PDT Fatty acid transport proteins (FATPs) provide pivotal roles in fatty acid transport and activation and thus are crucial for overall fatty acid homeostasis. Peroxisome Proliferators Activated Receptors (PPARs) are important transcription factors, which control many genes that govern lipid metabolism.

Using 293 T-REx cell lines that stably express FATP1 or FATP4 from a tetracycline-inducible promoter, this work evaluated gene expression of key genes involved in fatty acid metabolism using QPCR, protein expression of FATP1 and FATP4 using Western blots, and fatty acid transport to address the roles of these two FATP isoforms in fatty acid homeostasis. This work found that the expression of FATP1 from the heterologous Tet promoter resulted in increased FATP4 expression from its native promoter; the expression of FATP4 from the Tet promoter did not, however, increase the expression of FATP1. These results identified an unexpected regulatory loop between FATP1 and FATP4, which is hypothesized to occur at the transcriptional or post transcriptional level. Western blots confirmed these relationships by evaluating the protein expression of FATP1 and FATP4. Studies monitoring the transport of fatty acids using the fluorescent long chain fatty acid C1-BODIPY-C12 demonstrated the expression of FATP1 resulted in higher levels of transport relative to FATP4 overexpression. To address whether this regulatory loop proceeds through PPARa or PPARg, the expression of these two genes along with PPARs target genes were monitored. The over expression of FATP1 and FATP4 from the Tet promoter did not increase PPARs gene expression; the expression of FATP1 from the Tet promoter did result in an increased expression of PPARa target genes. Collectively these results supported the conclusion that the enzymatic product of FATP1 resulted in increased expression of FATP4 through a PPARα-mediated process. To further address this question, fatty acid and fatty acyl CoA profiles were measured in 293 T-REx cells expressing FATP1 or FATP4. The acyl CoA profiles showed both C18:2-CoA and C20:4-CoA were elevated in FATP1 expressing cell lines, perhaps indicating a relationship in n-3 fatty acid activation including further downstream metabolism. Likely effectors in this regulatory loop are arachidonic acid metabolites, including prostaglandins.

Advisor: Paul N. Black

]]> Zhe Yuan Functional Studies of Human Cellular Detoxification Enzymes http://digitalcommons.unl.edu/biochemdiss/6 http://digitalcommons.unl.edu/biochemdiss/6 Wed, 20 Apr 2011 07:05:26 PDT Cellular detoxification allows for the maintenance of cellular homeostasis and prevention of abnormal cell growth by clearing harmful xenobiotics and endobiotics. After oxygenation by phase I enzymes, phase II enzymes such as glucuronosyltransferases and glutathione-s-transferases conjugate a small molecule to the compound, marking it for subsequent export. Many up-stream enzymes are also essential to cellular detoxification by supplying the small compounds for conjugation. These up-stream enzymes include UDP-glucose dehydrogenase, which synthesizes UDP-glucuronate, and glutamate cysteine ligase, which catalyzes the first and rate-limiting step in the synthesis of glutathione.
UDP-glucose dehydrogenase (UGDH) is an important enzyme in human development and in the progression of many types of human epithelial cancers. Recently, mutations in UGDH were identified that are associated with congenital heart defects and cause a shift from a hexameric to a dimeric state. These clinical mutants, along with two engineered dimer mutants were used to examine differences in UGDH function resulting from loss of hexameric structure. The dimer mutants exhibited near wild-type activity in vitro, and significant differences in UDP-glucuronate levels were not observed in HEK 293 cells. Despite this, the phenotype of development defects associated with the UGDH clinical mutants is at least partially explained by a reduction in protein stability.
Glutamate cysteine ligase (GCL) deficiency is a rare autosomal recessive trait that compromises production of glutathione, a critical redox buffer and enzymatic cofactor. Glutamate cysteine ligase is a heterodimer comprised of a catalytic (GCLC) and a regulatory subunit (GCLM). Four clinical missense mutations have been identified within GCLC: Arg127Cys, Pro158Leu, His370Leu, and Pro414Leu. Embryonic fibroblasts from GCLC null mice were transiently transfected with wild-type or mutant GCLC and cellular glutathione levels were determined to be significantly lower in the mutants relative to wild-type. In an S. cerevisiae model system, mutant GCLC alone could not complement a glutathione-deficient strain and required the concurrent addition of GCLM to restore growth. Kinetic characterizations of the recombinant GCLC mutants indicated that the Arg127Cys, His370Leu, and Pro414Leu mutants have compromised enzymatic activity that can largely be rescued by the addition of GCLM, while the Pro158Leu mutant has kinetic constants comparable to wild-type GCLC.

]]> Melanie Neely Willis The In Situ Function of a Microbial Community Profiled by FT-IR: A Snapshot in Time http://digitalcommons.unl.edu/biochemdiss/5 http://digitalcommons.unl.edu/biochemdiss/5 Fri, 03 Dec 2010 10:41:46 PST Photographs of an ecosystem are an important tool in macro-community ecology. The photograph is a permanent record of species phenotype. In microbiology, biochemical activity provides the most descriptive information of an organism’s phenotype. A method for fingerprinting all biochemical activities occurring within a microbial community is analogous to a photograph. Infrared spectroscopy in the region between wavelengths 2500 to 20,000 nm (mid-IR) is a well established instrumental method for fingerprinting the total biochemical profile of axenic cultures. Spectra are complex and sensitive as demonstrated by the ability to discriminate between strains of bacteria, fungi, and algae. This thesis develops the method to apply mid-IR spectroscopy in order to attain a biochemical fingerprint by attenuated total reflectance. Chapter 2 establishes methods for obtaining mid-IR spectra by prefiltering and concentrating lake water onto a 0.2 µm filter membrane. In order to optimize signal, a combined spectrum from PVDF and nylon membranes is used following a 20-25 µm prefilter. Chapter 3 verifies that the established method is capable of discriminating the biochemical fingerprints of six interdunal lakes from Western Nebraska with differing chemistries and community structures. Mid-IR spectra provide functional data, but do not mirror structure, based on 16S rRNA data, identically. Now, a record of biochemical activity within a microbial community can be captured for future comparison. This technique is culture-independent and provides functional, in situ information. In the future, more information can be deduced by comparing different functional communities to one another, assigning spectral information to particular biochemical activities in a community.

]]> Ryan Roberts METHIONINE-R-SULFOXIDE REDUCTASES AND BIOLOGICAL IMPORTANCE OF FREE METHIONINE SULFOXIDE REDUCTION http://digitalcommons.unl.edu/biochemdiss/4 http://digitalcommons.unl.edu/biochemdiss/4 Fri, 01 Oct 2010 12:21:00 PDT We identified and functionally characterized yeast fRMsr homolog which showed specificity for reduction of free Met-R-SO and contributed to oxidative stress resistance of yeast cells. We further identified three conserved Cys which participate in catalysis through disulfide exchange. Evolutionary analyses revealed that the occurrence of fRMSr is restricted to unicellular organisms. We further found that mammalian MsrA can reduce free Met-S-SO, whereas MsrBs do not reduce free Met-R-SO. Consistent with these findings and the lack of fRMsr, mammalian cells could not grow in media that replaced Met with Met-R-SO. However, they grew in the presence of free Met-S-SO, which was reduced by MsrA. Expression of yeast fRMsr in mammalian cells supported the growth of mammalian cells on Met-R-SO, increased resistamce to oxidative stress, and affected expression levels of proteins that are regulated by Met availability.

We extended the finding of deficiency in free Met-R-SO reduction in mammals to examine the ability of Msrs to reduce compounds containing methylsulfinyls. Methylsulfide group has a prochiral sulfur, resulting in two diastereomers upon sulfoxidation. We found that methylsulfinyls in sulmazole sulfoxide, triclabendazole sulfoxide, and mesoridazine could be stereospecifically reduced by MsrA, but MsrB and fRMsr were not active. We also found that dimethylsulfoxide (DMSO), which has a methylsulfinyl group, can only be reduced by MsrA.

To examine the role of Met in aging, we performed lifespan analyses in fruit flies. There was no difference in lifespan of flies maintained on restricted (0.1 mM Met) and regular (1 mM) Met diets. Instead, we observed uncoupling between reproduction and longevity. A popular theory of aging suggests that dietary restriction is inversely proportional to reproduction due to nutritional redistribution of resources from egg generation to somatic maintenance, but we found that this theory did not follow our data that are based on variation in Met concentration.

Overall, our studies characterized all three known types of methionine sulfoxide reductase and uncovered biological significance of free methionine sulfoxide reduction and its deficiency in mammals.

]]> Byung Cheon Lee Consequences of Hyaluronan Metabolism on Cell Cycle Progression in Prostate Tumor Growth and Metastasis http://digitalcommons.unl.edu/biochemdiss/3 http://digitalcommons.unl.edu/biochemdiss/3 Tue, 21 Apr 2009 07:47:30 PDT Progression of Prostate cancer (CaP), depends on a complex series of interactions between the tumor and extracellular matrix (ECM) leading to tumor growth and metastasis. Hyaluronan (HA), an ECM component, is elevated in CaP and its accumulation in the tumor microenvironment is dependent on its synthetic enzyme, hyaluronan synthase (HAS), and turnover enzyme, hyaluronidase (Hyal). Although Hyal1 expression can independently prognosticate CaP, and HAS expression shows increase in aggressive prostate cancer cells, the functional relevance of this correlation is unexplored. In these studies we aim to dissect the roles of HAS and Hyal1 in prostate cancer.

Stably overexpressed hyaluronan synthases (HAS2, HAS3), and Hyal1, individually or concurrently (Hyal1+HAS2 and Hyal1+HAS3) in a non-metastatic prostate tumor cell line, were characterized by subcutaneous and orthotopic injections in NOD/SCID mice and assayed for cell proliferation, adhesion, and motility in vitro. Subcutaneous tumors were larger for Hyal1+HAS2 cells and smaller in Hyal1+HAS3 cells, potentially due to effective HA dissipation in the former. With orthoptopic injections, Hyal1+HAS transfectants were maximally tumorigenic and metastatic. Contrastingly, HAS expression suppressed tumor growth and metastasis, while Hyal1 promoted metastasis without significant tumor growth. These in vitro characterizations suggested tumor suppression by HAS was due to reduced growth with slower cell cycle kinetics, and the enhanced metastasis of Hyal1+HAS ascribed to increased adhesion and motility of the transfectants.

We tested ERK activation and cell cycle proteins as potential targets and found that Hyal1 showed modestly decreased sustained ERK activation compared to HAS3. Furthermore, in asynchronous and cycling cells, cyclin-dependent kinase inhibitors (CKIs) p21cipM and p27kip were elevated with HAS expression while p21cip expression was reduced in Hyal1 and Hyal1+HAS3 cells.

We demonstrate the dual requirement of both HAS and Hyal for maximum prostate tumorgenesis and metastasis mediated via altered adhesion, motility, and cell cycle progression. Our studies are the first comprehensive examination of the role of HAS and Hyal1 in prostate tumor progression thereby establishing a new paradigm for HA metabolism in CaP.

]]> Alamelu G. Bharadwaj Identification and Characterization of a Cadmium-Transporting P-Type ATPase in Yeast <i>Saccharomyces cerevisiae</i> http://digitalcommons.unl.edu/biochemdiss/2 http://digitalcommons.unl.edu/biochemdiss/2 Wed, 03 Dec 2008 14:01:52 PST Detoxification and homeostatic acquisition of metal ions are vital for all living organisms. Non-physiological heavy metals are toxic at low concentrations and represent major environmental hazards to human health. In particular, cadmium is a toxic environmental pollutant linked to a number of ailments including cancer, kidney and bone disease and reproductive disorders. The biological effects of cadmium toxicity which lead to human disease and the cellular mechanisms for cadmium defense are ill defined. Thus, the study of heavy metal detoxification systems represents an important research avenue to help combat cadmium related disorders.

The goal of this research project was to identify novel factors involved in metal ion defense in the model eukaryote, Saccharomyces cerevisiae. A genetic screen led to the discovery of Pca1, a P-type ATPase which functions as a cadmium-specific efflux pump. Unexpectedly, the PCA1 allele in common laboratory yeast strains possesses an inactivating mis-sense mutation which has complicated previous attempts to characterize its function.

A unique feature of Pca1 is a cysteine-rich cytosolic amino terminal extension that can participate in metal coordination and regulate expression levels. Under normal conditions, Pca1 is an unstable protein that is ubiquitinated and rapidly degraded by the proteasome. However, in the presence of cadmium, ubiquitination is inhibited resulting in rapid up-regulation and trafficking of Pca1 to the cell surface for cadmium efflux. An autonomous degradation signal within the cysteine-rich domain is necessary and sufficient for metal responsive regulation.

Finally, a second genetic screen was devised to uncover factors involved in Pca1 degradation. Unexpectedly, components of the ER-associated degradation (ERAD) pathway were found to be required for Pca1 turnover in the absence of cadmium. Conformational changes associated with cadmium binding to the metal sensing degradation signal lead to the escape of Pca1 from ERAD.

In summary, our data have revealed a novel metal detoxification system in a eukaryotic organism mediated by a P-type ATPase that is unique in structure and substrate specificity. Furthermore, we have described an unprecedented mode of ligand regulated degradation of a cell surface transporter at the ER and have provided the mechanistic basis of this regulation.

]]> David J. Adle Structural Basis of Allosteric and Intrasteric Regulation in Human Cystathionine β-Synthase and its Regulation by a CXXC Motif http://digitalcommons.unl.edu/biochemdiss/1 http://digitalcommons.unl.edu/biochemdiss/1 Wed, 01 Aug 2007 13:16:24 PDT The human cystathionine β-synthase (CBS) is a 5’ pyridoxal phosphate (PLP) dependent protein that catalyzes the condensation reaction between serine and homocysteine to yield cystathionine. This is the first step in the transsulfuration pathway which connects the methionine cycle to cysteine production. Mutations in CBS are the single most common cause of severe hereditary hyperhomocystenemia and over one hundred pathogenic mutations have been described so far. These mutations represent residues which are important for the structural and functional integrity of the enzyme. The biochemical characterization of these mutants thus leads to further insights into structure-function correlations in CBS.

CBS has a subunit mass of 63 kDa and is a modular protein with 551 amino acids. The N-terminal half of the protein houses the two cofactors, PLP and heme and a CXXC motif comprised of residues C272 P273, G274 and C275. This motif is also found in the thioredoxin family of proteins where it is involved in thiol disulfide exchange reactions. The heme, which is coordinated by the axial ligands H65 and C52, is about 20 Å away from the CXXC motif as well as the PLP catalytic center. As such, heme does not contribute directly to catalysis, but is believed to be a center for redox regulation in CBS.

The C-terminal half of the protein comprising residues 413 to 551 bear a tandem repeat of “CBS domains,” which are secondary structural motifs found in diverse families of proteins with no sequence similarities. In CBS, these domains are believed to be involved in the binding of the allosteric activator S-adenosyl methionine (AdoMet). Along with being involved in allosteric regulation, the C-terminal domain of CBS imposes intrasteric regulation on the catalytic core. Thus deletion of the C-terminal regulatory domain leads to the formation of a super-active enzyme, CBS-ΔC143 which has a 4-fold higher kcat than the full-length protein. It is also known that a truncated form of CBS is generated in cells exposed to proinflamatory agents such as TNFα. Although this observation suggests that the C-terminal domain interacts with the N-terminal catalytic core to effect this regulation, so far no experimental results were available which would characterize the nature of this interaction. In other words no information was available regarding the conformational changes associated with the allosteric and intrasteric regulation in CBS.

In this study, we have mapped the regions of intrasteric and allosteric regulation in CBS. We have employed hydrogen/deuterium (H/D) exchange, mass spectrometric technique to locate conformational changes in the enzyme upon the binding of its allosteric activator, AdoMet. We have also used this approach to detect the surface involved in the interaction of the C-terminal domain with the catalytic core relevant to intrasteric regulation. A change in the kinetics of H/D exchange was located in a single peptide, extending from residues 511-531 upon AdoMet binding. CBS-D444N a patient mutant exhibits a similar change in the above peptide in its native state and thus samples a conformation which is acquired by the wild type upon AdoMet binding. Accordingly this mutant is not responsive to any further activation by the allosteric activator. Peptides 356-370 and 371-385 in the N-terminal half of the protein exhibited a change in H/D exchange kinetics when the full-length and its counter part in CBS-ΔC143, were compared by H/D exchange studies, associating this region with intrasteric regulation. We have also demonstrated that in addition to heme, the CXXC motif in CBS is a center for redox regulation. Thiol alkylation followed by mass spectrometric analysis demonstrated formation of an intramolecular disulfide bridge between C272 and C275, and identified the presence of a sulfenic acid intermediate under air oxidized conditions in CBS-ΔC143. The full length and the CBS-ΔC143 enzyme exhibited a 1.6 and 4.5 fold enhancement in specific activity respectively upon reduction of their disulfides at the CXXC center. A redox potential of -240 ± 4 mV was determined for the CXXC center using MAL-PEG for titrating thiol content at various ratios of oxidized and reduced dithiotreitol.

Advisor: Ruma Banerjee

]]> Suvajit Sen