Substitution of active site tyrosines with tryptophan alters the free energy for nucleotide flipping by human alkyladenine DNA glycosylase.
Jenna Hendershot, with her mentor Professor Patrick O'Brien, and recent graduate Dr. Abigail Wolfe collaborated on a paper for Biochemistry and had some surprising results.
Human alkyladenine DNA glycosylase (AAG) locates and excises a wide variety of structurally diverse alkylated and oxidized purine lesions from DNA to initiate the base excision repair pathway. Recognition of a base lesion requires flipping of the damaged nucleotide into a relatively open active site pocket between two conserved tyrosine residues, Y127 and Y159. We have mutated each of these amino acids to tryptophan and measured the kinetic effects on the nucleotide flipping and base excision steps. The Y127W and Y159W mutant proteins have robust glycosylase activity toward DNA containing 1,N(6)-ethenoadenine (εA), within 4-fold of that of the wild-type enzyme, raising the possibility that tryptophan fluorescence could be used to probe the DNA binding and nucleotide flipping steps. Stopped-flow fluorescence was used to compare the time-dependent changes in tryptophan fluorescence and εA fluorescence. For both mutants, the tryptophan fluorescence exhibited two-step binding with essentially identical rate constants as were observed for the εA fluorescence changes. These results provide evidence that AAG forms an initial recognition complex in which the active site pocket is perturbed and the stacking of the damaged base is disrupted. Upon complete nucleotide flipping, there is further quenching of the tryptophan fluorescence with coincident quenching of the εA fluorescence. Although these mutations do not have large effects on the rate constant for excision of εA, there are dramatic effects on the rate constants for nucleotide flipping that result in 40-100-fold decreases in the flipping equilibrium relative to wild-type. Most of this effect is due to an increased rate of unflipping, but surprisingly the Y159W mutation causes a 5-fold increase in the rate constant for flipping. The large effect on the equilibrium for nucleotide flipping explains the greater deleterious effects that these mutations have on the glycosylase activity toward base lesions that are in more stable base pairs. PubMed
Hendershot JM, Wolfe AE, O'Brien PJ.
Biochemistry. 2011 Mar 22;50(11):1864-74. Epub 2011 Feb 3.
Joseph Micucci, a student in Dan Bochar's lab, was cited at JBC as a Paper of the Week. His paper, Regulated Cleavage of Prothrombin by Prothrombinase, was published in the January, 2010 issue of Journal of Biological Chemistry and is subtitled "Repositioning a cleavage site reveals the unique kinetic behavior of the action of Prothrombinase on its compound substrate." It can be read at JBC.
A University of Michigan and University of Oxford collaboration recently earned front page attention in the Royal Society of Chemistry’s journal
Read more on the team’s work, including its earlier coverage in the Journal of the American Chemical Society.
Stacie Bulfer, of Ray Trievel's lab, presented her thesis defense on February 5. Her paper, Structural basis for L-lysine feedback inhibition of homocitrate synthase, was published in in the April, 2010 issue of the Journal of Biological Chemistry, and was cited as a JBC Paper of the Week. Papers of the Week represent the top 1% of papers reviewed in terms of significance and overall importance at JBC. Her paper can be read at JBC.
Graduate student Chase Weidmann is listed listed as first author on Drosophila Pumilio Protein Contains Multiple Autonomous Repression Domains That Regulate mRNAs Independently of Nanos and Brain Tumor, written with his mentor Aaron Goldstrohm. Their findings suggest that PUF proteins have evolved new regulatory functions through protein sequences appended to their conserved PUF repeat RNA-binding domains. Read the abstract for this Molecular and Cellular Biology paper here PMID: 22064486
Having noticed that Myelin-derived inhibitors limit axon outgrowth and plasticity during development in the adult mammalian central nervous system, BioChem's Professors David Turner and Anne Vojtek have collaborated with Graduate Student Heather Dickson on her first published paper in a recent issue of the Journal of Neuroscience. The entire paper may be read at J. Neuro
Valentin Cracan, is a student in Ruma Banerjee's lab, current interest is the elucidation of a newly discovered radical B12 enzyme, isobutyryl-CoA mutase, which is fused to a GTP-dependent chaperone, His paper, IcmF Is a Fusion between the Radical B12 Enzyme Isobutyryl-CoA Mutase and Its G-protein Chaperone, was published in the October, 2009 issue of the Journal of Biological Chemistry. It can be read at JBC.
Ryan Evans, is a student in Audrey Seasholtz's lab. His research recognizes that CRH directs the physiological and behavioral responses to stress. Its activity is mediated by CRH receptors (CRH-R) 1 and 2 and modulated by the CRH-binding protein. Aberrant regulation of this system has been associated with anxiety disorders and major depression, demonstrating the importance of understanding the regulation of CRH activity. The paper, Soluble Corticotropin-Releasing Hormone Receptor 2Î± Splice Variant is Efficiently Translated, but not Trafficked for Secretion., was published in October, 2009. It can be read at Endocrinology.
Sean Ferris, is a student in Randal J. Kaufman's lab, where they are interested in protein folding and misfolding in cells, specifically in the endoplasmic reticulum (ER). A cellular demand for protein exceeding the protein-folding capacity of the ER results in a situation known as ER stress. His paper, Generation and phenotyping of mCd59a and mCd59b double-knockout mice, was published in February, 2009 issue of American Journal of Hematology. For it, see AJH.
Jennifer Gehret, is a student in Janet Smith's lab. Her paper, Polyketide Decarboxylative Chain Termination Preceded by O-Sulfonation in Curacin A Biosynthesis, was published in the October, 2009 issue of the Journal of the American Chemical Society. It describes how biosynthetic innovation in natural product systems is driven by the recruitment of new genes and enzymes into these complex pathways. It can be read at JACS.
Nirupama Gupta, is a student in Stephen Ragsdale's lab. Gupta is interested in how Cysteine synthase (Cys11) plays a dual role in maintaining the correct structure for DNA binding and in redox regulation of CprK. Her paper, Dual Roles of an Essential Cysteine Residue in Activity of a Redox-regulated Bacterial Transcriptional Activator, was published in the October, 2008 issue of the Journal of Biological Chemistry. It can be read at JBC.
Justin Hassler, a student in Randal Kaufman's lab, discusses in a paper findings that suggest an unprecedented link by which protein synthesis and/or misfolding in the ER causes oxidative stress and should encourage the development of novel strategies to treat diabetes. The paper, The unfolded protein response is required to maintain the integrity of the endoplasmic reticulum, prevent oxidative stress and preserve differentiation in Î²-cells., was published in the October, 2010 issue of the Diabetes, Obesity and Metabolism. It can be read at DOM.
Corissa Lamphear, is a student in Carol Fierke's lab, where her interest is how a better understanding of the molecular recognition of substrates performed by FTase will aid in both designing new FTase inhibitors as therapeutic agents and characterizing proteins involved in prenylation-dependent cellular pathways. Her paper, Context-Dependent Substrate Recognition by Protein Farnesyltransferaseâ€ , was published in the February, 2009 issue of the Biochemistry. It can be read at Biochemistry.
Li Li, is a student in Kun-Liang Guan's lab. Her paper, Rheb controls misfolded protein metabolism by inhibiting aggresome formation and autophagy, was published in the May, 2009 issue of the Proceedings of the National Academy of Sciences. This study reveals a function of Rheb in controlling misfolded protein metabolism by modulating aggresome formation. It can be read at PNAS.
Thiol-disulfide redox dependence of heme binding and heme ligand switching in a nuclear hormone receptor Rev-erbβ
Rev-erbβ is a heme-binding nuclear hormone receptor that represses a broad spectrum of target genes involved in regulating metabolism, the circadian cycle and proinflammatory responses. Graduate Student Nirupama Gupta has collaborated with Professor Steve Ragdale on a paper in recent issue of the Journal of Biological Chemistry. They suggest that this thiol-disulfide redox switch is one mechanism by which oxidative stress is linked to circadian and/or metabolic imbalance; and propose that oxidative stress leads to oxidation of cysteine(s), thus releasing heme from Rev-erbβ and altering its transcriptional activity. JBC
Identification and Characterization of Oxalate Oxidoreductase, a Novel Thiamine Pyrophosphate-dependent 2-Oxoacid Oxidoreductase
Moorella thermoacetica is an anaerobic acetogen, a class of bacteria that is found in the soil, the animal gastrointestinal tract, and the rumen. This organism engages the Wood-Ljungdahl pathway of anaerobic CO(2) fixation for heterotrophic or autotrophic growth. Graduate Student Elizabeth Pierce has collaborated with Professor Steve Ragdale on a paper in a recent issue of the Journal of Biological Chemistry. It describes a novel enzyme, oxalate oxidoreductase (OOR), that enables M. thermoacetica to grow on oxalate, which is produced in soil and is a common component of kidney stones. The entire paper may be read at JBC
iRhoms are inactive rhomboid-like pseudoproteases that lack essential catalytic residues. Although iRhoms are highly conserved in metazoan species, little is known about their function. In a recent issue of Developmental Cell, Graduate Student Stewart Siyan Cao and collaborator Professor Randal J. Kaufman discuss recent insights on how iRhoms regulate growth factors signaling through endoplasmic reticulum-associated protein degradation (ERAD.
Highly conserved among all sequenced metazoans, iRhoms are related to rhomboid intramembrane serine proteases, but are not active proteases, as they lack the essential catalytic residues required for serine proteases and have no proteolytic activity. Although their high degree of conservation between organisms suggests that these pseudoproteases are under evolutionary selective pressure, their functional significance is largely unknown. A previous study reported that human iRhom1 interacts with the human epidermal growth factor (EGF) when it is overexpressed in Drosophila, suggesting a possible link between an iRhom and EGFR signaling in mammals (Freeman, 2008). In a recent issue of Cell, Zettl et al. (2011) show in both Drosophila and mammalian cells that iRhoms regulate growth factor signaling (e.g., EGFR pathways) through the ER quality control machinery that function in endoplasmic reticulum-associated protein degradation (ERAD)
Cao SS, Kaufman RJ.
Comment on Cell. 2011 Apr 1;145(1):79-91.
The entire paper may be read at PubMed
The recent issue of Biochemistry features Loss of allostery and coenzyme B12 delivery by a pathogenic mutation in adenosyltransferase, a paper co-authored by graduate student Michael Lofgren and Professor Ruma Banerjee.
ATP-dependent cob(I)alamin adenosyltransferase (ATR) is a bifunctional protein: an enzyme that catalyzes the adenosylation of cob(I)alamin and an escort that delivers the product, adenosylcobalamin (AdoCbl or coenzyme B(12)), to methylmalonyl-CoA mutase (MCM), resulting in holoenzyme formation. Failure to assemble holo-MCM leads to methylmalonic aciduria. We have previously demonstrated that only 2 equiv of AdoCbl bind per homotrimer of ATR and that binding of ATP to the vacant active site triggers ejection of 1 equiv of AdoCbl from an adjacent site. In this study, we have mimicked in the Methylobacterium extorquens ATR, a C-terminal truncation mutation, D180X, described in a patient with methylmalonic aciduria, and characterized the associated biochemical penalties. We demonstrate that while k(cat) and K(M)(Cob(I)) for D180X ATR are only modestly decreased (by 3- and 2-fold, respectively), affinity for the product, AdoCbl, is significantly diminished (400-fold), and the negative cooperativity associated with its binding is lost. We also demonstrate that the D180X mutation corrupts ATP-dependent cofactor ejection, which leads to transfer of AdoCbl from wild-type ATR to MCM. These results suggest that the pathogenicity of the corresponding human truncation mutant results from its inability to sequester AdoCbl for direct transfer to MCM. Instead, cofactor release into solution is predicted to reduce the capacity for holo-MCM formation, leading to disease.
Lofgren M, Banerjee R.
Biochemistry. 2011 Jun 28;50(25):5790-8. Epub 2011 Jun 2.
The entire paper may be read at PubMed