A5046973248- Enzyme Structure and Function
- Amoebic Infections and Treatments
- Biochemical and Molecular Research
- Microbial Metabolic Engineering and Bioproduction
- Protein Structure and Dynamics
- Pediatric Hepatobiliary Diseases and Treatments
- Enzyme Production and Characterization
- Anaerobic Digestion and Biogas Production
- Biofuel production and bioconversion
- Genomics and Phylogenetic Studies
- Cancer, Hypoxia, and Metabolism
- Parasitic Infections and Diagnostics
- Microbial Natural Products and Biosynthesis
- Nutrition, Genetics, and Disease
- Legume Nitrogen Fixing Symbiosis
- Enzyme function and inhibition
- Endoplasmic Reticulum Stress and Disease
- Pancreatitis Pathology and Treatment
- Polyamine Metabolism and Applications
- Plant nutrient uptake and metabolism
- Enzyme Catalysis and Immobilization
- Photosynthetic Processes and Mechanisms
- Heat shock proteins research
- Polysaccharides and Plant Cell Walls
- Essential Oils and Antimicrobial Activity
Clemson University
2009-2023
Pennsylvania State University
1997-2005
Massachusetts Institute of Technology
2002
University of Wisconsin–Madison
2002
University of Illinois Urbana-Champaign
2002
Purdue University West Lafayette
2001
Virginia Tech
1998
Methanogenesis, the biological production of methane, plays a pivotal role in global carbon cycle and contributes significantly to warming. The majority methane nature is derived from acetate. Here we report complete genome sequence an acetate-utilizing methanogen, Methanosarcina acetivorans C2A. Methanosarcineae are most metabolically diverse methanogens, thrive broad range environments, unique among Archaea forming complex multicellular structures. This diversity reflected M. . At...
ABSTRACT Acetate kinase, an enzyme widely distributed in the Bacteria and Archaea domains, catalyzes phosphorylation of acetate. We have determined three-dimensional structure Methanosarcina thermophila acetate kinase bound to ADP through crystallography. As we previously predicted, contains a core fold that is topologically identical ADP-binding domains glycerol hexokinase, 70-kDa heat shock cognate (Hsc70), actin. Numerous charged active-site residues are conserved within kinases, but few...
The genome sequence of the aceticlastic methanoarchaeon Methanosaeta concilii GP6, comprised a 3,008,626-bp chromosome and an 18,019-bp episome, has been determined exhibits considerable differences in gene content from that thermophila.
Chlamydomonas reinhardtii insertion mutants disrupted for genes encoding acetate kinases (EC 2.7.2.1) (ACK1 and ACK2) a phosphate acetyltransferase 2.3.1.8) (PAT2, but not PAT1) were isolated to characterize fermentative production. ACK1 PAT2 localized chloroplasts, while ACK2 PAT1 shown be in mitochondria. Characterization of the showed that activity chloroplasts plays dominant role (relative mitochondria) producing under dark, anoxic conditions and, surprisingly, also suggested has other...
Entamoeba histolytica is a parasitic protozoan that causes amoebic dysentery, which affects approximately 90 million people each year worldwide. E. transmitted through ingestion of food and water contaminated with the cyst form, undergoes excystation in small intestine to trophozoite form colonizes large intestine. The reptile pathogen invadens has served as model for studying stage conversion between due lack reproducible encystation laboratory. Although much been learned about using...
AMP-forming acetyl-CoA synthetase [ACS; acetate:CoA ligase (AMP-forming), EC 6.2.1.1] catalyzes the activation of acetate to in a two-step reaction. This enzyme is member adenylate-forming superfamily that includes firefly luciferase, nonribosomal peptide synthetases, and acyl- aryl-CoA synthetases/ligases. Although structures several members demonstrate these enzymes have similar fold domain structure, low sequence conservation diversity substrates utilized limited utility understanding...
ABSTRACT Acetate kinase catalyzes the reversible magnesium-dependent synthesis of acetyl phosphate by transfer ATP γ-phosphoryl group to acetate. Inspection crystal structure Methanosarcina thermophila enzyme containing only ADP revealed a solvent-accessible hydrophobic pocket formed residues Val 93 , Leu 122 Phe 179 and Pro 232 in active site cleft, which identified potential acetate binding site. The hypothesis that this was further supported alignment all sequences available from...
Adenosine monophosphate (AMP)‐forming acetyl‐CoA synthetase (ACS; acetate:CoA ligase (AMP‐forming), EC 6.2.1.1) is a key enzyme for conversion of acetate to acetyl‐CoA, an essential intermediate at the junction anabolic and catabolic pathways. Phylogenetic analysis putative short medium chain acyl‐CoA sequences indicates that ACSs form distinct clade from other synthetases. Within this clade, archaeal are not monophyletic fall into three groups composed both bacterial sequences. Kinetic two...
The roles of an aspartate and arginine, which are completely conserved in the active sites beta-class carbonic anhydrases, were investigated by steady-state kinetic analyses replacement variants enzyme (Cab) from archaeon Methanobacterium thermoautotrophicum. Previous wild-type Cab indicated a two-step zinc-hydroxide mechanism catalysis k(cat)/K(m) value depends only on rate constants for CO(2) hydration step, whereas k(cat) also proton transfer step (K. S. Smith, N. J. Cosper, C....
ABSTRACT Acetate kinase catalyzes the reversible phosphorylation of acetate (CH 3 COO − + ATP⇄CH CO 2 PO 2− ADP). A mechanism which involves a covalent phosphoryl-enzyme intermediate has been proposed, and chemical modification studies enzyme from Escherichia coli indicate an unspecified glutamate residue is phosphorylated (J. A. Todhunter D. L. Purich, Biochem. Biophys. Res. Commun. 60:273–280, 1974). Alignment amino acid sequences for kinases E. ( Bacteria domain), Methanosarcina...
Site-directed mutagenesis is a powerful tool for identifying active-site residues essential catalysis; however, this approach has only recently become available acetate kinase. The enzyme from Methanosarcina thermophila been cloned and hyper-produced in highly active form Escherichia coli (recombinant wild-type). role of arginines kinase was investigated. Five (R91, R175, R241, R285, R340) the M. were selected individual replacement based on their high conservation among sequences...
ABSTRACT Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp), previously thought to be present only in bacteria but recently found fungi, catalyzes the formation of acetyl phosphate from xylulose 5-phosphate or fructose 6-phosphate. Here, we describe first biochemical and kinetic characterization a eukaryotic Xfp, opportunistic fungal pathogen Cryptococcus neoformans , which has two XFP genes (designated XFP1 XFP2 ). Our C. Xfp2 indicated existence both substrate cooperativity...
The role of histidine in the catalytic mechanism acetate kinase from Methanosarcina thermophila was investigated by diethylpyrocarbonate inactivation and site-directed mutagenesis. Inactivation accompanied an increase absorbance at 240 nm with no change 280 nm, treatment inactivated enzyme hydroxylamine restored 95% activity, results that indicated inactivates specific modification histidine. substrates ATP, ADP, acetate, acetyl phosphate protected against suggesting least one active site...
Abstract The acyl‐AMP forming family of adenylating enzymes catalyze two‐step reactions to activate a carboxylate with the chemical energy derived from ATP hydrolysis. X‐ray crystal structures have been determined for multiple members this and, together biochemical studies, provide insights into active site and catalytic mechanisms used by these enzymes. These studies shown that use domain rotation 140° reconfigure single two partial reactions. We present here structure new medium chain...
Acetate kinase (ACK) catalyzes the reversible synthesis of acetyl phosphate by transfer γ-phosphate ATP to acetate. Here we report first biochemical and kinetic characterization a eukaryotic ACK, that from protist Entamoeba histolytica. Our revealed this ACK is only known member ASKHA structural superfamily, which includes acetate kinase, hexokinase, other sugar kinases, utilize inorganic pyrophosphate (PP(i))/inorganic (P(i)) as sole phosphoryl donor/acceptor. Detection activity in E....
Entamoeba histolytica, an amitochondriate protozoan parasite that relies on glycolysis as a key pathway for ATP generation, has developed unique extended PPi-dependent glycolytic in which ADP-forming acetyl-coenzyme A (CoA) synthetase (ACD; acetate:CoA ligase [ADP-forming]; EC 6.2.1.13) converts acetyl-CoA to acetate produce additional and recycle CoA. We characterized the recombinant E. histolytica ACD found enzyme is bidirectional, allowing it potentially play role production or...
ABSTRACT Short- and medium-chain acyl coenzyme A (acyl-CoA) synthetases catalyze the formation of acyl-CoA from an substrate, ATP, CoA. These enzymes mechanistically similar two-step reactions that proceed through enzyme-bound acyl-AMP intermediate. Here we describe characterization a member this enzyme family methane-producing archaeon Methanosarcina acetivorans . This enzyme, synthetase designated Macs Ma , utilizes 2-methylbutyrate as its preferred substrate for synthesis but cannot...
Abstract The unique biochemical properties of acetate kinase present a classic conundrum in the study mechanism enzyme‐catalyzed phosphoryl transfer. Large, single crystals from Methanosarcina thermophila were grown solution ammonium sulfate presence ATP. diffract to beyond 1.7 Å resolution. Analysis X‐ray data is consistent with space group C 2 and unit cell dimensions = 181 Å, b 67 c 83 β 103°. Diffraction have been collected at 110 277 K. Data K extend lower resolution, but are more...
ABSTRACT Periplasmic cyclic β-glucans of Rhizobium species provide important functions during plant infection and hypo-osmotic adaptation. In Sinorhizobium meliloti (also known as ), these molecules are highly modified with phosphoglycerol succinyl substituents. We have previously identified an S. Tn 5 insertion mutant, S9, which is specifically impaired in its ability to transfer substituents the β-glucan backbone (M. W. Breedveld, J. A. Hadley, K. Miller, Bacteriol. 177:6346–6351, 1995)....
Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for reversible phosphorylation to acetyl phosphate utilizing ATP as substrate. kinases are ubiquitous in Bacteria, found one genus Archaea, also present microbes Eukarya6. The most well characterized kinase that from methane-producing archaeon Methanosarcina thermophila7-14. An which can only utilize PPi but not phosphate-forming direction has been isolated Entamoeba...
ABSTRACT The cyclic β-(1,2)-glucans of Rhizobium meliloti and Agrobacterium tumefaciens play an important role during hypoosmotic adaptation, the synthesis these compounds is osmoregulated. Glucosyltransferase, enzyme responsible for β-(1,2)-glucan biosynthesis, present constitutively, suggesting that osmotic regulation biosynthesis glucans occurs through modulation activity. In this study, we examined glucan in vitro with membrane preparations from R. . results show ionic solutes inhibit...
The acyl-adenylate-forming enzyme superfamily, consisting of acyl- and aryl-CoA synthetases, the adenylation domain nonribosomal peptide luciferase, has three signature motifs (I–III) ten conserved core (A1–A10), some which overlap motifs. consensus sequence for motif III (core A7) in acetyl-CoA synthetase is Y-X-S/T/A-G-D, with an invariant fifth position, highly first fourth positions, variable second third positions. Kinetic studies variants revealed that alteration at any position...