Cheryl Ingram-Smith

Bio: Cheryl Ingram-Smith is an academic researcher from Pennsylvania State University. The author has contributed to research in topics: Acetate kinase & Methanosarcina thermophila. The author has an hindex of 9, co-authored 12 publications receiving 980 citations. Previous affiliations of Cheryl Ingram-Smith include University of Illinois at Urbana–Champaign & Washington University in St. Louis.

The Genome of M. Acetivorans Reveals Extensive Metabolic and Physiological Diversity

Abstract: The Archaea remain the most poorly understood domain of life despite their importance to the biosphere. Methanogenesis, which plays a pivotal role in the global carbon cycle, is unique to the Archaea. Each year, an estimated 900 million metric tons of methane are biologically produced, representing the major global source for this greenhouse gas and contributing significantly to global warming (Schlesinger 1997). Methanogenesis is critical to the waste-treatment industry and biologically produced methane also represents an important alternative fuel source. At least two-thirds of the methane in nature is derived from acetate, although only two genera of methanogens are known to be capable of utilizing this substrate. We report here the first complete genome sequence of an acetate-utilizing (acetoclastic) methanogen, Methanosarcina acetivorans C2A. The Methanosarcineae are metabolically and physiologically the most versatile methanogens. Only Methanosarcina species possess all three known pathways for methanogenesis (Fig. ​(Fig.1)1) and are capable of utilizing no less than nine methanogenic substrates, including acetate. In contrast, all other orders of methanogens possess a single pathway for methanogenesis, and many utilize no more than two substrates. Among methanogens, the Methanosarcineae also display extensive environmental diversity. Individual species of Methanosarcina have been found in freshwater and marine sediments, decaying leaves and garden soils, oil wells, sewage and animal waste digesters and lagoons, thermophilic digesters, feces of herbivorous animals, and the rumens of ungulates (Zinder 1993). Figure 1 Three pathways for methanogenesis. Methanogenesis is a form of anaerobic respiration using a variety of one-carbon (C-1) compounds or acetic acid as a terminal electron acceptor. All three pathways converge on the reduction of methyl-CoM to methane (CH ... The Methanosarcineae are unique among the Archaea in forming complex multicellular structures during different phases of growth and in response to environmental change (Fig. ​(Fig.2).2). Within the Methanosarcineae, a number of distinct morphological forms have been characterized, including single cells with and without a cell envelope, as well as multicellular packets and lamina (Macario and Conway de Macario 2001). Packets and lamina display internal morphological heterogeneity, suggesting the possibility of cellular differentiation. Moreover, it has been suggested that cells within lamina may display differential production of extracellular material, a potential form of cellular specialization (Macario and Conway de Macario 2001). The formation of multicellular structures has been proposed to act as an adaptation to stress and likely plays a role in the ability of Methanosarcina species to colonize diverse environments. Figure 2 Different morphological forms of Methanosarcina acetivorans. Thin-section electron micrographs showing M. acetivorans growing as both single cells (center of micrograph) and within multicellular aggregates (top left, bottom right). Cells were harvested ... Significantly, powerful methods for genetic analysis exist for Methanosarcina species. These tools include plasmid shuttle vectors (Metcalf et al. 1997), very high efficiency transformation (Metcalf et al. 1997), random in vivo transposon mutagenesis (Zhang et al. 2000), directed mutagenesis of specific genes (Zhang et al. 2000), multiple selectable markers (Boccazzi et al. 2000), reporter gene fusions (M. Pritchett and W. Metcalf, unpubl.), integration vectors (Conway de Macario et al. 1996), and anaerobic incubators for large-scale growth of methanogens on solid media (Metcalf et al. 1998). Furthermore, and in contrast to other known methanogens, genetic analysis can be used to study the process of methanogenesis: Because Methanosarcina species are able to utilize each of the three known methanogenic pathways, mutants in a single pathway are viable (M. Pritchett and W. Metcalf, unpubl.). The availability of genetic methods allowing immediate exploitation of genomic sequence, coupled with the genetic, physiological, and environmental diversity of M. acetivorans make this species an outstanding model organism for the study of archaeal biology. For these reasons, we set out to study the genome of M. acetivorans.

Urkinase: Structure of Acetate Kinase, a Member of the ASKHA Superfamily of Phosphotransferases

Abstract: Acetate kinase, an enzyme widely distributed in theBacteria and Archaea domains, catalyzes the phosphorylation of acetate. We have determined the three-dimensional structure of Methanosarcina thermophila acetate kinase bound to ADP through crystallography. As we previously predicted, acetate kinase contains a core fold that is topologically identical to that of the ADP-binding domains of glycerol kinase, hexokinase, the 70-kDa heat shock cognate (Hsc70), and actin. Numerous charged active-site residues are conserved within acetate kinases, but few are conserved within the phosphotransferase superfamily. The identity of the points of insertion of polypeptide segments into the core fold of the superfamily members indicates that the insertions existed in the common ancestor of the phosphotransferases. Another remarkable shared feature is the unusual, epsilon conformation of the residue that directly precedes a conserved glycine residue (Gly-331 in acetate kinase) that binds the α-phosphate of ADP. Structural, biochemical, and geochemical considerations indicate that an acetate kinase may be the ancestral enzyme of the ASKHA (acetate and sugar kinases/Hsc70/actin) superfamily of phosphotransferases.

Characterization of the Acetate Binding Pocket in the Methanosarcina thermophila Acetate Kinase

Abstract: Acetate kinase catalyzes the reversible magnesium-dependent synthesis of acetyl phosphate by transfer of the ATP γ-phosphoryl group to acetate. Inspection of the crystal structure of the Methanosarcina thermophila enzyme containing only ADP revealed a solvent-accessible hydrophobic pocket formed by residues Val93, Leu122, Phe179, and Pro232 in the active site cleft, which identified a potential acetate binding site. The hypothesis that this was a binding site was further supported by alignment of all acetate kinase sequences available from databases, which showed strict conservation of all four residues, and the recent crystal structure of the M. thermophila enzyme with acetate bound in this pocket. Replacement of each residue in the pocket produced variants with Km values for acetate that were 7- to 26-fold greater than that of the wild type, and perturbations of this binding pocket also altered the specificity for longer-chain carboxylic acids and acetyl phosphate. The kinetic analyses of variants combined with structural modeling indicated that the pocket has roles in binding the methyl group of acetate, influencing substrate specificity, and orienting the carboxyl group. The kinetic analyses also indicated that binding of acetyl phosphate is more dependent on interactions of the phosphate group with an unidentified residue than on interactions between the methyl group and the hydrophobic pocket. The analyses also indicated that Phe179 is essential for catalysis, possibly for domain closure. Alignments of acetate kinase, propionate kinase, and butyrate kinase sequences obtained from databases suggested that these enzymes have similar catalytic mechanisms and carboxylic acid substrate binding sites.

Identification of Essential Glutamates in the Acetate Kinase from Methanosarcina thermophila

Abstract: Acetate kinase catalyzes the reversible phosphorylation of acetate (CH3COO- + ATP CH3CO2PO3(2-) + ADP). A mechanism which involves a covalent phosphoryl-enzyme intermediate has been proposed, and chemical modification studies of the enzyme from Escherichia coli indicate an unspecified glutamate residue is phosphorylated (J. A. Todhunter and D. L. Purich, Biochem. Biophys. Res. Commun. 60:273-280, 1974). Alignment of the amino acid sequences for the acetate kinases from E. coli (Bacteria domain), Methanosarcina thermophila (Archaea domain), and four other phylogenetically divergent microbes revealed high identity which included five glutamates. These glutamates were replaced in the M. thermophila enzyme to determine if any are essential for catalysis. The histidine-tagged altered enzymes were produced in E. coli and purified to electrophoretic homogeneity by metal affinity chromatography. Replacements of E384 resulted in either undetectable or extremely low kinase activity, suggesting E384 is essential for catalysis which supports the proposed mechanism. Replacement of E385 influenced the Km values for acetate and ATP with only moderate decreases in k(cat), which suggests that this residue is involved in substrate binding but not catalysis. The unaltered acetate kinase was not inactivated by N-ethylmaleimide; however, replacement of E385 with cysteine conferred sensitivity to N-ethylmaleimide which was prevented by preincubation with acetate, acetyl phosphate, ATP, or ADP, suggesting that E385 is located near the active site. Replacement of E97 decreased the Km value for acetate but not ATP, suggesting this residue is involved in binding acetate. Replacement of either E32 or E334 had no significant effects on the kinetic constants, which indicates that neither residue is essential for catalysis or significantly influences the binding of acetate or ATP.

Roles of the Conserved Aspartate and Arginine in the Catalytic Mechanism of an Archaeal β-Class Carbonic Anhydrase

Abstract: The roles of an aspartate and an arginine, which are completely conserved in the active sites of β-class carbonic anhydrases, were investigated by steady-state kinetic analyses of replacement variants of the β-class enzyme (Cab) from the archaeon Methanobacterium thermoautotrophicum. Previous kinetic analyses of wild-type Cab indicated a two-step zinc-hydroxide mechanism of catalysis in which the k cat / K m value depends only on the rate constants for the CO 2 hydration step, whereas k cat also depends on rate constants from the proton transfer step (K. S. Smith, N. J. Cosper, C. Stalhandske, R. A. Scott, and J. G. Ferry, J. Bacteriol. 182: 6605-6613, 2000). The recently solved crystal structure of Cab shows the presence of a buffer molecule within hydrogen bonding distance of Asp-34, implying a role for this residue in the proton transport step (P. Strop, K. S. Smith, T. M. Iverson, J. G. Ferry, and D. C. Rees, J. Biol. Chem. 276: 10299-10305, 2001). The k cat / K m values of Asp-34 variants were decreased relative to those of the wild type, although not to an extent which supports an essential role for this residue in the CO 2 hydration step. Parallel decreases in k cat and k cat / K m values for the variants precluded any conclusions regarding a role for Asp-34 in the proton transfer step; however, the k cat of the D34A variant was chemically rescued by replacement of 2-( N -morpholino)propanesulfonic acid buffer with imidazole at pH 7.2, supporting a role for the conserved aspartate in the proton transfer step. The crystal structure of Cab also shows Arg-36 with two hydrogen bonds to Asp-34. Arg-36 variants had both k cat and k cat / K m values that were decreased at least 250-fold relative to those of the wild type, establishing an essential function for this residue. Imidazole was unable to rescue the k cat of the R36A variant; however, partial rescue of the kinetic parameter was obtained with guanidine-HCl indicating that the guanido group of this residue is important.

Methanogenic archaea: ecologically relevant differences in energy conservation.

Abstract: Most methanogenic archaea can reduce CO(2) with H(2) to methane, and it is generally assumed that the reactions and mechanisms of energy conservation that are involved are largely the same in all methanogens. However, this does not take into account the fact that methanogens with cytochromes have considerably higher growth yields and threshold concentrations for H(2) than methanogens without cytochromes. These and other differences can be explained by the proposal outlined in this Review that in methanogens with cytochromes, the first and last steps in methanogenesis from CO(2) are coupled chemiosmotically, whereas in methanogens without cytochromes, these steps are energetically coupled by a cytoplasmic enzyme complex that mediates flavin-based electron bifurcation.

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

Abstract: The phosphoenolpyruvate(PEP):carbohydrate phosphotransferase system (PTS) is found only in bacteria, where it catalyzes the transport and phosphorylation of numerous monosaccharides, disaccharides, amino sugars, polyols, and other sugar derivatives. To carry out its catalytic function in sugar transport and phosphorylation, the PTS uses PEP as an energy source and phosphoryl donor. The phosphoryl group of PEP is usually transferred via four distinct proteins (domains) to the transported sugar bound to the respective membrane component(s) (EIIC and EIID) of the PTS. The organization of the PTS as a four-step phosphoryl transfer system, in which all P derivatives exhibit similar energy (phosphorylation occurs at histidyl or cysteyl residues), is surprising, as a single protein (or domain) coupling energy transfer and sugar phosphorylation would be sufficient for PTS function. A possible explanation for the complexity of the PTS was provided by the discovery that the PTS also carries out numerous regulatory functions. Depending on their phosphorylation state, the four proteins (domains) forming the PTS phosphorylation cascade (EI, HPr, EIIA, and EIIB) can phosphorylate or interact with numerous non-PTS proteins and thereby regulate their activity. In addition, in certain bacteria, one of the PTS components (HPr) is phosphorylated by ATP at a seryl residue, which increases the complexity of PTS-mediated regulation. In this review, we try to summarize the known protein phosphorylation-related regulatory functions of the PTS. As we shall see, the PTS regulation network not only controls carbohydrate uptake and metabolism but also interferes with the utilization of nitrogen and phosphorus and the virulence of certain pathogens.

The acetate switch.

Abstract: To succeed, many cells must alternate between life-styles that permit rapid growth in the presence of abundant nutrients and ones that enhance survival in the absence of those nutrients. One such change in life-style, the “acetate switch,” occurs as cells deplete their environment of acetate-producing carbon sources and begin to rely on their ability to scavenge for acetate. This review explains why, when, and how cells excrete or dissimilate acetate. The central components of the “switch” (phosphotransacetylase [PTA], acetate kinase [ACK], and AMP-forming acetyl coenzyme A synthetase [AMP-ACS]) and the behavior of cells that lack these components are introduced. Acetyl phosphate (acetyl∼P), the high-energy intermediate of acetate dissimilation, is discussed, and conditions that influence its intracellular concentration are described. Evidence is provided that acetyl∼P influences cellular processes from organelle biogenesis to cell cycle regulation and from biofilm development to pathogenesis. The merits of each mechanism proposed to explain the interaction of acetyl∼P with two-component signal transduction pathways are addressed. A short list of enzymes that generate acetyl∼P by PTA-ACKA-independent mechanisms is introduced and discussed briefly. Attention is then directed to the mechanisms used by cells to “flip the switch,” the induction and activation of the acetate-scavenging AMP-ACS. First, evidence is presented that nucleoid proteins orchestrate a progression of distinct nucleoprotein complexes to ensure proper transcription of its gene. Next, the way in which cells regulate AMP-ACS activity through reversible acetylation is described. Finally, the “acetate switch” as it exists in selected eubacteria, archaea, and eukaryotes, including humans, is described.

Metabolic, phylogenetic, and ecological diversity of the methanogenic archaea.

Abstract: Although of limited metabolic diversity, methanogenic archaea or methanogens possess great phylogenetic and ecological diversity. Only three types of methanogenic pathways are known: CO(2)-reduction, methyl-group reduction, and the aceticlastic reaction. Cultured methanogens are grouped into five orders based upon their phylogeny and phenotypic properties. In addition, uncultured methanogens that may represent new orders are present in many environments. The ecology of methanogens highlights their complex interactions with other anaerobes and the physical and chemical factors controlling their function.

The general transcription machinery and general cofactors.

Abstract: In eukaryotes, the core promoter serves as a platform for the assembly of transcription preinitiation complex (PIC) that includes TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and RNA polymerase II (pol II), which function collectively to specify the transcription start site. PIC formation usually begins with TFIID binding to the TATA box, initiator, and/or downstream promoter element (DPE) found in most core promoters, followed by the entry of other general transcription factors (GTFs) and pol II through either a sequential assembly or a preassembled pol II holoenzyme pathway. Formation of this promoter-bound complex is sufficient for a basal level of transcription. However, for activator-dependent (or regulated) transcription, general cofactors are often required to transmit regulatory signals between gene-specific activators and the general transcription machinery. Three classes of general cofactors, including TBP-associated factors (TAFs), Mediator, and upstream stimulatory activity (USA)-derived positive cofactors (PC1/PARP-1, PC2, PC3/DNA topoisomerase I, and PC4) and negative cofactor 1 (NC1/HMGB1), normally function independently or in combination to fine-tune the promoter activity in a gene-specific or cell-type-specific manner. In addition, other cofactors, such as TAF1, BTAF1, and negative cofactor 2 (NC2), can also modulate TBP or TFIID binding to the core promoter. In general, these cofactors are capable of repressing basal transcription when activators are absent and stimulating transcription in the presence of activators. Here we review the roles of these cofactors and GTFs, as well as TBP-related factors (TRFs), TAF-containing complexes (TFTC, SAGA, SLIK/SALSA, STAGA, and PRC1) and TAF variants, in pol II-mediated transcription, with emphasis on the events occurring after the chromatin has been remodeled but prior to the formation of the first phosphodiester bond.