John C. Taylor

The Bifunctional Active Site of S-Adenosylmethionine Synthetase ROLES OF THE ACTIVE SITE ASPARTATES

S-Adenosylmethionine (AdoMet) synthetase catalyzes the biosynthesis of AdoMet in a unique enzymatic reaction. Initially the sulfur of methionine displaces the intact tripolyphosphate chain (PPPi) from ATP, and subsequently PPPi is hydrolyzed to PPi and Pibefore product release. The crystal structure of Escherichia coli AdoMet synthetase shows that the active site contains four aspartate residues. Aspartate residues Asp-16* and Asp-271 individually provide the sole protein ligand to one of the two required Mg2+ ions (* denotes a residue from a second subunit); aspartates Asp-118 and Asp-238* are proposed to interact with methionine. Each aspartate has been changed to an uncharged asparagine, and the metal binding residues were also changed to alanine, to assess the roles of charge and ligation ability on catalytic efficiency. The resultant enzyme variants all structurally resemble the wild type enzyme as indicated by circular dichroism spectra and are tetramers. However, all have k cat reductions of ∼103-fold in AdoMet synthesis, whereas the MgATP and methionine K m values change by less than 3- and 8-fold, respectively. In the partial reaction of PPPihydrolysis, mutants of the Mg2+ binding residues have >700-fold reduced catalytic efficiency (k cat/K m ), whereas the D118N and D238*N mutants are impaired less than 35-fold. The catalytic efficiency for PPPi hydrolysis by Mg2+ site mutants is improved by AdoMet, like the wild type enzyme. In contrast AdoMet reduces the catalytic efficiency for PPPi hydrolysis by the D118N and D238*N mutants, indicating that the events involved in AdoMet activation are hindered in these methionyl binding site mutants. Ca2+ uniquely activates the D271A mutant enzyme to 15% of the level of Mg2+, in contrast to the ∼1% Ca2+ activation of the wild type enzyme. This indicates that the Asp-271 side chain size is a discriminator between the activating ability of Ca2+ and the smaller Mg2+.

Monovalent Cation Activation and Kinetic Mechanism of Inosine 5′-Monophosphate Dehydrogenase

Human type II inosine 5′-monophosphate dehydrogenase has been purified to homogeneity from an Escherichia coli strain that express large quantities of the enzyme from the cloned gene. Steady state kinetic studies have been used to characterize the activation by monovalent cations, including Li, Na, K, Rb, Cs, Tl, NH, and N(CH). The enzyme has less than 1% of the maximal activity in the absence of an added monovalent cation, such as K, Na, Rb, Tl, or NH. The enzyme is activated by K and Tl at lower concentrations than those of other monovalent cations. Li and N(CH) do not activate the enzyme, nor do they inhibit the K-activated enzyme, implying that ionic radius is important in binding selectivity. The K values for both substrates and V differ with different monovalent cations. Initial velocity and product inhibition kinetic data are consistent with an ordered steady state mechanism in which the enzyme binds K first, IMP second, and then NAD; the product NADH is released before xanthosine 5′-monophosphate. Substrate and product binding experiments support this mechanism and show the presence of one substrate binding site per subunit. Several rate constants were obtained from a computer simulation of the complete steady state rate equation.

The Bifunctional Active Site of S-Adenosylmethionine Synthetase ROLES OF THE BASIC RESIDUES

S-Adenosylmethionine (AdoMet) synthetase catalyzes a unique two-step enzymatic reaction leading to formation of the primary biological alkylating agent. The crystal structure of Escherichia coli AdoMet synthetase shows that the active site, which lies between two subunits, contains four lysines and one histidine as basic residues. In order to test the proposed charge and hydrogen bonding roles in catalytic function, each lysine has been changed to an uncharged methionine or alanine, and the histidine has been altered to asparagine. The resultant enzyme variants are all tetramers like the wild type enzyme; however, circular dichroism spectra show reductions in helix content for the K245*M and K269M mutants. (The asterisk denotes that the residue is in the second subunit.) Four mutants have k cat reductions of ∼103–104-fold in AdoMet synthesis; however, the k cat of K165*M variant is only reduced 2-fold. In each mutant, there is a smaller catalytic impairment in the partial reaction of tripolyphosphate hydrolysis. The K165*A enzyme has a 100-fold greater k cat for tripolyphosphate hydrolysis than the wild type enzyme, but this mutant is not activated by AdoMet in contrast to the wild type enzyme. The properties of these mutants require reassessment of the catalytic roles of these residues.

Isozymes of S-adenosylmethionine synthetase are encoded by tandemly duplicated genes in Escherichia coli

The sole biosynthetic route to S‐adenosylmethionine, the primary biological alkylating agent, is catalysed by S‐adenosylmethionine synthetase (ATP: L‐methionine S‐adenosyltransferase). In Escherichia coli and Sal‐monella typhimunum numerous studies have located a structural gene (metK) for this enzyme at 63min on the chromosomal map. We have now identified a second structural gene for S‐adenosylmethionine synthetase in E. coli by DNA hybridization experiments with metK as the probe; we denote this gene as metX. The metX gene is located adjacent to metK with the gene order speA metK metX speC. The metK and metX genes are separated by ∼0.8kb. The metK and the metX genes are oriented convergently as indicated by DNA hybridization experiments using sequences from the 5′ and 3′ ends of metK. The metK gene product is detected immunochemically only in cells growing in minimal media, whereas the metX gene product is detected immunochemically in cells grown in rich media at all growth phases and in stationary phase in minimal media.

Subunit association as the stabilizing determinant for archaeal methionine adenosyltransferases

Archaea contain a class of methionine adenosyltransferases (MATs) that exhibit substantially higher stability than their mesophilic counterparts. Their sequences are highly divergent, but preserve the essential active site motifs of the family. We have investigated the origin of this increased stability using chemical denaturation experiments on Methanococcus jannaschii MAT (Mj-MAT) and mutants containing single tryptophans in place of tyrosine residues. The results from fluorescence, circular dichroism, hydrodynamic, and enzyme activity measurements showed that the higher stability of Mj-MAT derives largely from a tighter association of its subunits in the dimer. Local fluorescence changes, interpreted using secondary structure predictions, further identify the least stable structural elements as the C-terminal ends of beta-strands E2 and E6, and the N-terminus of E3. Dimer dissociation however requires a wider perturbation of the molecule. Additional analysis was initially hindered by the lack of crystal structures for archaeal MATs, a limitation that we overcame by construction of a 3D-homology model of Mj-MAT. This model predicts preservation of the chain topology and three-domain organization typical of this family, locates the least stable structural elements at the flat contact surface between monomers, and shows that alterations in all three domains are required for dimer dissociation.

A New Role for ERα: Silencing via DNA Methylation of Basal, Stem Cell, and EMT Genes

Resistance to hormonal therapies is a major clinical problem in the treatment of estrogen receptor α–positive (ERα+) breast cancers. Epigenetic marks, namely DNA methylation of cytosine at specific CpG sites (5mCpG), are frequently associated with ERα+ status in human breast cancers. Therefore, ERα may regulate gene expression in part via DNA methylation. This hypothesis was evaluated using a panel of breast cancer cell line models of antiestrogen resistance. Microarray gene expression profiling was used to identify genes normally silenced in ERα+ cells but derepressed upon exposure to the demethylating agent decitabine, derepressed upon long-term loss of ERα expression, and resuppressed by gain of ERα activity/expression. ERα-dependent DNA methylation targets (n = 39) were enriched for ERα-binding sites, basal-up/luminal-down markers, cancer stem cell, epithelial–mesenchymal transition, and inflammatory and tumor suppressor genes. Kaplan–Meier survival curve and Cox proportional hazards regression analyses indicated that these targets predicted poor distant metastasis–free survival among a large cohort of breast cancer patients. The basal breast cancer subtype markers LCN2 and IFI27 showed the greatest inverse relationship with ERα expression/activity and contain ERα-binding sites. Thus, genes that are methylated in an ERα-dependent manner may serve as predictive biomarkers in breast cancer. Implications: ERα directs DNA methylation–mediated silencing of specific genes that have biomarker potential in breast cancer subtypes. Mol Cancer Res; 15(2); 152–64. ©2016 AACR.

Structural basis for the stability of a thermophilic methionine adenosyltransferase against guanidinium chloride

The methionine adenosyltransferase from the thermophile Methanococcus jannaschii is fully and irreversibly unfolded in the presence of guanidinium chloride. Unfolding of this dimeric protein is a three-state process in which a dimeric intermediate could be identified. The less stable secondary structural elements of the protein are the C-terminal ends of β-strands E2 and E6, as deduced from the behavior of tyrosine to tryptophan mutants at residues 72 and 170, which are located in the subunit interface. Unraveling of these elements at the monomer interface may soften intersubunit interactions, leading to the observed 85% activity loss. Accumulation of the intermediate was associated with maintenance of residual activity, an increase in the elution volume of the protein upon gel filtration and a decrease in the sedimentation coefficient. Elimination of the remaining enzymatic activity occurred in conjunction with a 50% reduction in helicity and fluorescence alterations illustrating a transient burial of tryptophans at β-strands E2, E3 and E9. The available 3D-model predicted that these β-strands are involved in the central and N-terminal domains of the monomer structure. Severe perturbation of this area of the monomer–monomer interface may destroy the remaining intermolecular interactions, thus leading to dissociation and aggregation. Finally, transition to the denatured state includes completion of the changes detected in the microenvironments around tryptophans included at α-helixes H5 and H6, the loops connecting H5–E8 and E9, β-strands E3 and E12.

The ORF1 of the gentamicin‐resistance operon (aac) of Pseudomonas aeruginosa encodes adenosine 5′‐phosphosulphate kinase

The gentamicin‐resistance operon of Pseudomonas aeruginosa (aac) contains two cistrons for which only the second gene product has an identified function. The 813bp second cistron (ORF2) encodes a protein that confers gentamicin resistance by catalysis of the transfer of an acetyl group from acetyl Coenzyme A to gentamicin. The first open reading frame (ORF1) encodes a 23.9 kDa protein that we have found, by enzyme activity and immunological reactivity, to be adenosine‐5′‐phosphosulphate (APS) kinase. APS kinase catalyses the transfer of the gamma phosphoryl group of ATP to the 3′‐hydroxyl group of APS. The 70% sequence similarity between the Pseudomonas and Escherichia coli APS kinases suggests that the Pseudomonas enzyme may catalyse phosphoryl transfer to the 3′‐hydroxyl group of other nucleotides such as dephosphocoenzyme A, as does the purified E. coli APS kinase. In extracts of pseudomonad cells we have also detected a higher molecular mass (70 kDa) protein that cross‐reacts with an anti‐E. coli APS kinase antibody. This cross‐reactive protein is also present in Pseudomonas strains lacking the gentamicin‐resistance plasmid, and apparently reflects an APS kinase analogous to the nodQ‐encoded high‐molecular‐weight APS kinase present in Rhizobium meliloti. Production of the Pseudomonas aac APS kinase was repressed by cysteine when expressed in E. coli, as is E. coli APS kinase. However, cysteine did not repress production of the Pseudomonas enzyme when the aac ORF1 ‐encoded enzyme was expressed in a Pseudomonas strain, indicating differential regulation of gene expression in the two organisms.

Abstract LB-152: A new role for ERα: Gene silencing via DNA methylation

As a master transcription factor, estrogen receptor-α (ERα) regulates expression of a multitude of genes. We hypothesized that ERα may also regulate gene expression via DNA methylation since methylation of specific CpG sites associates with ERα-positive status in human breast cancer. We tested this hypothesis by identifying genes normally silenced in ERα-positive T47D and MCF-7 luminal breast cancer cell lines but which became expressed (or derepressed) upon exposure to the demethylating agent decitabine and antagonizing ERα activity. These genes were identified using a combination of Agilent gene expression microarrays and assessed according to the Significance Analysis of Microarrays (SAM) method at a 2-fold change and 5% FDR. Selected genes were validated by pyrosequencing to quantitate CpG methylation, lentiviral re-introduction of ERα, real-time PCR and immunoblotting. In a short-term study, T47D cells were treated with decitabine and the ER antagonist fulvestrant (FUL) for 96 h. Array analysis identified 87 genes that were derepressed by decitabine and FUL, of which 31 (36%) were markers of the basal subtype of breast cancer. In long-term studies (8 - 36 weeks), MCF-7 and T47D cells were FUL-treated or estrogen-deprived (ED) to derive multiple antihormone-resistant cell lines (MCF-7/FUL, T47D/FUL, and 2 independent T47D/ED), all of which lost ≥ 95% of ERα. Comparing all of the resistant cell lines and decitabine-treated T47D cells by array analysis revealed that 58 genes were up-regulated by both loss of ERα and decitabine treatment, 35 (60%) of which were basal markers. Two basal breast cancer subtype markers, LCN2 and IFI27 which are involved in epithelial-mesenchymal transition and interferon signaling, respectively, were selected for verification. First, LCN2 and IFI27 expression increased ∼132- and 343-fold, respectively, while their CpG methylation levels significantly decreased upon ERα loss in all T47D antihormone-resistant cell lines. Second, LCN2 and IFI27 expression decreased while their methylation levels increased upon estrogen re-exposure or lentiviral ERα re-introduction in the T47D/ED cell lines. Moreover, high CpG methylation levels of LCN2 and IFI27 directly associated with ERα-positive status but inversely correlated with their expression levels across a panel of 12 breast cancer cell lines. Therefore, ERα targets specific genes, such as basal markers, for DNA methylation-mediated silencing. Mechanistically, ERα-dependent DNA methylation may occur as a consequence of long-term transcriptional repression. Although not well appreciated, estrogen-bound ERα represses transcription of more genes than it activates. Thus, ERα may direct DNA methylation by interacting with multicomponent corepressor complexes, which could then recruit DNA methyltransferases to target promoters. Moreover, ERα-dependent silencing of basal markers may promote the prognostically more favorable luminal breast cancer subtype. Citation Format: Eric A. Ariazi, John C. Taylor, Emmanuelle Nicolas, Michael J. Slifker, Jeff Boyd. A new role for ERα: Gene silencing via DNA methylation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-152. doi:10.1158/1538-7445.AM2015-LB-152