PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-15 (15)
 

Clipboard (0)
None

Select a Filter Below

Journals
Year of Publication
1.  Biochemical and Kinetic Characterization of Xylulose 5-Phosphate/Fructose 6-Phosphate Phosphoketolase 2 (Xfp2) from Cryptococcus neoformans 
Eukaryotic Cell  2014;13(5):657-663.
Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp), previously thought to be present only in bacteria but recently found in fungi, catalyzes the formation of acetyl phosphate from xylulose 5-phosphate or fructose 6-phosphate. Here, we describe the first biochemical and kinetic characterization of a eukaryotic Xfp, from the opportunistic fungal pathogen Cryptococcus neoformans, which has two XFP genes (designated XFP1 and XFP2). Our kinetic characterization of C. neoformans Xfp2 indicated the existence of both substrate cooperativity for all three substrates and allosteric regulation through the binding of effector molecules at sites separate from the active site. Prior to this study, Xfp enzymes from two bacterial genera had been characterized and were determined to follow Michaelis-Menten kinetics. C. neoformans Xfp2 is inhibited by ATP, phosphoenolpyruvate (PEP), and oxaloacetic acid (OAA) and activated by AMP. ATP is the strongest inhibitor, with a half-maximal inhibitory concentration (IC50) of 0.6 mM. PEP and OAA were found to share the same or have overlapping allosteric binding sites, while ATP binds at a separate site. AMP acts as a very potent activator; as little as 20 μM AMP is capable of increasing Xfp2 activity by 24.8% ± 1.0% (mean ± standard error of the mean), while 50 μM prevented inhibition caused by 0.6 mM ATP. AMP and PEP/OAA operated independently, with AMP activating Xfp2 and PEP/OAA inhibiting the activated enzyme. This study provides valuable insight into the metabolic role of Xfp within fungi, specifically the fungal pathogen Cryptococcus neoformans, and suggests that at least some Xfps display substrate cooperative binding and allosteric regulation.
doi:10.1128/EC.00055-14
PMCID: PMC4060483  PMID: 24659577
2.  Crystal Structures of Acetate Kinases from the Eukaryotic Pathogens Entamoeba histolytica and Cryptococcus neoformans 
Journal of structural biology  2012;181(2):185-189.
Acetate kinases (ACKs) are members of the acetate and sugar kinase/hsp70/actin (ASKHA) superfamily and catalyze the reversible phosphorylation of acetate, with ADP/ATP the most common phosphoryl acceptor/donor. While prokaryotic ACKs have been the subject of extensive biochemical and structural characterization, there is a comparative paucity of information on eukaryotic ACKs, and prior to this report, no structure of an ACK of eukaryotic origin was available. We determined the structures of ACKs from the eukaryotic pathogens Entamoeba histolytica and Cryptococcus neoformans. Each active site is located at an interdomain interface, and the acetate and phosphate binding pockets display sequence and structural conservation with their prokaryotic counterparts. Interestingly, the E. histolytica ACK has previously been shown to be pyrophosphate (PPi)-dependent, and is the first ACK demonstrated to have this property. Examination of its structure demonstrates how subtle amino acid substitutions within the active site have converted cosubstrate specificity from ATP to PPi while retaining a similar backbone conformation. Differences in the angle between domains surrounding the active site suggest that interdomain movement may accompany catalysis. Taken together, these structures are consistent with the eukaryotic ACKs following a similar reaction mechanism as is proposed for the prokaryotic homologs.
doi:10.1016/j.jsb.2012.11.001
PMCID: PMC3565045  PMID: 23159802
Acetate kinase; PPi-dependent kinase; ASKHA superfamily
3.  Harvest for Health Gardening Intervention Feasibility Study in Cancer Survivors 
Acta oncologica (Stockholm, Sweden)  2013;52(6):1110-1118.
Background
Cancer survivors are at increased risk for second malignancies, cardiovascular disease, diabetes, and functional decline. Evidence suggests that a healthful diet and physical activity may reduce the risk of chronic disease and improve health in this population.
Methods
We conducted a feasibility study to evaluate a vegetable gardening intervention that paired 12 adult and child cancer survivors with Master Gardeners to explore effects on fruit and vegetable intake, physical activity, quality-of-life, and physical function. Throughout the year-long study period, the survivor-Master Gardener dyads worked together to plan/plant 3 gardens, harvest/rotate plantings, and troubleshoot/correct problems. Data on diet, physical activity, and quality-of-life were collected via surveys; anthropometrics and physical function were objectively measured. Acceptability of the intervention was assessed with a structured debriefing survey.
Results
The gardening intervention was feasible (robust enrollment; minimal attrition) and well-received by cancer survivors and Master Gardeners. Improvement in 3 of 4 objective measures of strength, agility, and endurance was observed in 90% of survivors, with the following change scores (median [interquartile range]) noted between baseline and 1-year follow-up: hand grip test (+4.8 [3.0, 6.7] kg), 8 foot Get-Up-and-Go (−1.0 [−1.8, −0.2] seconds), 30-second chair stand (+3.0 [−1.0, 5.0] stands), and 6-minute walk (+38 [20, 160] feet). Increases of ≥1 fruit and vegetable serving/ day and ≥30 minutes/week of physical activity were observed in 40% and 60%, respectively.
Conclusion
These preliminary results support the feasibility and acceptability of a mentored gardening intervention and suggest that it may offer a novel and promising strategy to improve fruit and vegetable consumption, physical activity, and physical function in cancer survivors. A larger randomized controlled trial is needed to confirm our results.
doi:10.3109/0284186X.2013.770165
PMCID: PMC3718632  PMID: 23438359
cancer survivors; gardening; intervention; health; diet
4.  Novel Pyrophosphate-Forming Acetate Kinase from the Protist Entamoeba histolytica 
Eukaryotic Cell  2012;11(10):1249-1256.
Acetate kinase (ACK) catalyzes the reversible synthesis of acetyl phosphate by transfer of the γ-phosphate of ATP to acetate. Here we report the first biochemical and kinetic characterization of a eukaryotic ACK, that from the protist Entamoeba histolytica. Our characterization revealed that this protist ACK is the only known member of the ASKHA structural superfamily, which includes acetate kinase, hexokinase, and other sugar kinases, to utilize inorganic pyrophosphate (PPi)/inorganic phosphate (Pi) as the sole phosphoryl donor/acceptor. Detection of ACK activity in E. histolytica cell extracts in the direction of acetate/PPi formation but not in the direction of acetyl phosphate/Pi formation suggests that the physiological direction of the reaction is toward acetate/PPi production. Kinetic parameters determined for each direction of the reaction are consistent with this observation. The E. histolytica PPi-forming ACK follows a sequential mechanism, supporting a direct in-line phosphoryl transfer mechanism as previously reported for the well-characterized Methanosarcina thermophila ATP-dependent ACK. Characterizations of enzyme variants altered in the putative acetate/acetyl phosphate binding pocket suggested that acetyl phosphate binding is not mediated solely through a hydrophobic interaction but also through the phosphoryl group, as for the M. thermophila ACK. However, there are key differences in the roles of certain active site residues between the two enzymes. The absence of known ACK partner enzymes raises the possibility that ACK is part of a novel pathway in Entamoeba.
doi:10.1128/EC.00169-12
PMCID: PMC3485911  PMID: 22903977
5.  Role of Motif III in Catalysis by Acetyl-CoA Synthetase 
Archaea  2012;2012:509579.
The acyl-adenylate-forming enzyme superfamily, consisting of acyl- and aryl-CoA synthetases, the adenylation domain of the nonribosomal peptide synthetases, and luciferase, has three signature motifs (I–III) and ten conserved core motifs (A1–A10), some of which overlap the signature motifs. The consensus sequence for signature motif III (core motif A7) in acetyl-CoA synthetase is Y-X-S/T/A-G-D, with an invariant fifth position, highly conserved first and fourth positions, and variable second and third positions. Kinetic studies of enzyme variants revealed that an alteration at any position resulted in a strong decrease in the catalytic rate, although the most deleterious effects were observed when the first or fifth positions were changed. Structural modeling suggests that the highly conserved Tyr in the first position plays a key role in active site architecture through interaction with a highly conserved active-site Gln, and the invariant Asp in the fifth position plays a critical role in ATP binding and catalysis through interaction with the 2′- and 3′-OH groups of the ribose moiety. Interactions between these Asp and ATP are observed in all structures available for members of the superfamily, consistent with a critical role in substrate binding and catalysis for this invariant residue.
doi:10.1155/2012/509579
PMCID: PMC3438747  PMID: 22973162
6.  Complete Genome Sequence of Methanosaeta concilii, a Specialist in Aceticlastic Methanogenesis ▿ 
Journal of Bacteriology  2011;193(14):3668-3669.
The genome sequence of the aceticlastic methanoarchaeon Methanosaeta concilii GP6, comprised of a 3,008,626-bp chromosome and an 18,019-bp episome, has been determined and exhibits considerable differences in gene content from that of Methanosaeta thermophila.
doi:10.1128/JB.05031-11
PMCID: PMC3133334  PMID: 21571998
7.  Characterization of an Archaeal Medium-Chain Acyl Coenzyme A Synthetase from Methanosarcina acetivorans▿  
Journal of Bacteriology  2010;192(22):5982-5990.
Short- and medium-chain acyl coenzyme A (acyl-CoA) synthetases catalyze the formation of acyl-CoA from an acyl substrate, ATP, and CoA. These enzymes catalyze mechanistically similar two-step reactions that proceed through an enzyme-bound acyl-AMP intermediate. Here we describe the characterization of a member of this enzyme family from the methane-producing archaeon Methanosarcina acetivorans. This enzyme, a medium-chain acyl-CoA synthetase designated MacsMa, utilizes 2-methylbutyrate as its preferred substrate for acyl-CoA synthesis but cannot utilize acetate and thus cannot catalyze the first step of acetoclastic methanogenesis in M. acetivorans. When propionate or other less favorable acyl substrates, such as butyrate, 2-methylpropionate, or 2-methylvalerate, were utilized, the acyl-CoA was not produced or was produced at reduced levels. Instead, acyl-AMP and PPi were released in the absence of CoA, whereas in the presence of CoA, the intermediate was broken down into AMP and the acyl substrate, which were released along with PPi. These results suggest that although acyl-CoA synthetases may have the ability to utilize a broad range of substrates for the acyl-adenylate-forming first step of the reaction, the intermediate may not be suitable for the thioester-forming second step. The MacsMa structure has revealed the putative acyl substrate- and CoA-binding pockets. Six residues proposed to form the acyl substrate-binding pocket, Lys256, Cys298, Gly351, Trp259, Trp237, and Trp254, were targeted for alteration. Characterization of the enzyme variants indicates that these six residues are critical in acyl substrate binding and catalysis, and even conservative alterations significantly reduced the catalytic ability of the enzyme.
doi:10.1128/JB.00600-10
PMCID: PMC2976453  PMID: 20851904
8.  The 2.1Å Crystal Structure of an Acyl-CoA Synthetase from Methanosarcina acetivorans reveals an alternate acyl binding pocket for small branched acyl substrates†,‡ 
Proteins  2009;77(3):685-698.
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 of this family and, together with biochemical studies, provide insights into the active site and catalytic mechanisms used by these enzymes. These studies have shown that the enzymes use a domain rotation of 140° to reconfigure a single active site to catalyze the two partial reactions. We present here the crystal structure of a new medium chain acyl-CoA synthetase from Methanosarcina acetivorans. The binding pocket for the three substrates is analyzed, with many conserved residues present in the AMP binding pocket. The CoA binding pocket is compared to the pockets of both acetyl-CoA synthetase and 4-chlorobenzoate:CoA ligase. Most interestingly, the acyl binding pocket of the new structure is compared with other acyl- and aryl-CoA synthetases. A comparison of the acyl-binding pocket of the acyl-CoA synthetase from M. acetivorans with other structures identifies a shallow pocket that is used to bind the medium chain carboxylates. These insights emphasize the high sequence and structural diversity among this family in the area of the acyl binding pocket.
doi:10.1002/prot.22482
PMCID: PMC2767475  PMID: 19544569
Adenylate-forming enzyme; substrate specificity; X-ray crystallography
9.  Activity of a Second Trypanosoma brucei Hexokinase Is Controlled by an 18-Amino-Acid C-Terminal Tail▿  
Eukaryotic Cell  2006;5(12):2014-2023.
Trypanosoma brucei expresses two hexokinases that are 98% identical, namely, TbHK1 and TbHK2. Homozygous null TbHK2−/− procyclic-form parasites exhibit an increased doubling time, a change in cell morphology, and, surprisingly, a twofold increase in cellular hexokinase activity. Recombinant TbHK1 enzymatic activity is similar to that of other hexokinases, with apparent Km values for glucose and ATP of 0.09 ± 0.02 mM and 0.28 ± 0.1 mM, respectively. The kcat value for TbHK1 is 2.9 × 104 min−1. TbHK1 can use mannose, fructose, 2-deoxyglucose, and glucosamine as substrates. In addition, TbHK1 is inhibited by fatty acids, with lauric, myristic, and palmitic acids being the most potent (with 50% inhibitory concentrations of 75.8, 78.4, and 62.4 μM, respectively). In contrast to TbHK1, recombinant TbHK2 lacks detectable enzymatic activity. Seven of the 10 amino acid differences between TbHK1 and TbHK2 lie within the C-terminal 18 amino acids of the polypeptides. Modeling of the proteins maps the C-terminal tails near the interdomain cleft of the enzyme that participates in the conformational change of the enzyme upon substrate binding. Replacing the last 18 amino acids of TbHK2 with the corresponding residues of TbHK1 yields an active recombinant protein with kinetic properties similar to those of TbHK1. Conversely, replacing the C-terminal tail of TbHK1 with the TbHK2 tail inactivates the enzyme. These findings suggest that the C-terminal tail of TbHK1 is important for hexokinase activity. The altered C-terminal tail of TbHK2, along with the phenotype of the knockout parasites, suggests a distinct function for the protein.
doi:10.1128/EC.00146-06
PMCID: PMC1694814  PMID: 17028241
10.  AMP-forming acetyl-CoA synthetases in Archaea show unexpected diversity in substrate utilization 
Archaea  2006;2(2):95-107.
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 of anabolic and catabolic pathways. Phylogenetic analysis of putative short and medium chain acyl-CoA synthetase sequences indicates that the ACSs form a distinct clade from other acyl-CoA synthetases. Within this clade, the archaeal ACSs are not monophyletic and fall into three groups composed of both bacterial and archaeal sequences. Kinetic analysis of two archaeal enzymes, an ACS from Methanothermobacter thermautotrophicus (designated as MT-ACS1) and an ACS from Archaeoglobus fulgidus (designated as AF-ACS2), revealed that these enzymes have very different properties. MT-ACS1 has nearly 11-fold higher affinity and 14-fold higher catalytic efficiency with acetate than with propionate, a property shared by most ACSs. However, AF-ACS2 has only 2.3-fold higher affinity and catalytic efficiency with acetate than with propionate. This enzyme has an affinity for propionate that is almost identical to that of MT-ACS1 for acetate and nearly tenfold higher than the affinity of MT-ACS1 for propionate. Furthermore, MT-ACS1 is limited to acetate and propionate as acyl substrates, whereas AF-ACS2 can also utilize longer straight and branched chain acyl substrates. Phylogenetic analysis, sequence alignment and structural modeling suggest a molecular basis for the altered substrate preference and expanded substrate range of AF-ACS2 versus MT-ACS1.
PMCID: PMC2686389  PMID: 17350930
acetate; Archaeoglobus fulgidus; Methanothermobacter thermautotrophicus
12.  Characterization of the Acetate Binding Pocket in the Methanosarcina thermophila Acetate Kinase 
Journal of Bacteriology  2005;187(7):2386-2394.
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.
doi:10.1128/JB.187.7.2386-2394.2005
PMCID: PMC1065240  PMID: 15774882
13.  Roles of the Conserved Aspartate and Arginine in the Catalytic Mechanism of an Archaeal β-Class Carbonic Anhydrase 
Journal of Bacteriology  2002;184(15):4240-4245.
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 kcat/Km value depends only on the rate constants for the CO2 hydration step, whereas kcat 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 kcat/Km 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 CO2 hydration step. Parallel decreases in kcat and kcat/Km values for the variants precluded any conclusions regarding a role for Asp-34 in the proton transfer step; however, the kcat 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 kcat and kcat/Km 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 kcat 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.
doi:10.1128/JB.184.15.4240-4245.2002
PMCID: PMC135220  PMID: 12107142
14.  Structural and Kinetic Characterization of an Archaeal β-Class Carbonic Anhydrase 
Journal of Bacteriology  2000;182(23):6605-6613.
The β-class carbonic anhydrase from the archaeon Methanobacterium thermoautotrophicum (Cab) was structurally and kinetically characterized. Analytical ultracentrifugation experiments show that Cab is a tetramer. Circular dichroism studies of Cab and the Spinacia oleracea (spinach) β-class carbonic anhydrase indicate that the secondary structure of the β-class enzymes is predominantly α-helical, unlike that of the α- or γ-class enzymes. Extended X-ray absorption fine structure results indicate the active zinc site of Cab is coordinated by two sulfur and two O/N ligands, with the possibility that one of the O/N ligands is derived from histidine and the other from water. Both the steady-state parameters kcat and kcat/Km for CO2 hydration are pH dependent. The steady-state parameter kcat is buffer-dependent in a saturable manner at both pH 8.5 and 6.5, and the analysis suggested a ping-pong mechanism in which buffer is the second substrate. At saturating buffer conditions and pH 8.5, kcat is 2.1-fold higher in H2O than in D2O, consistent with an intramolecular proton transfer step being rate contributing. The steady-state parameter kcat/Km is not dependent on buffer, and no solvent hydrogen isotope effect was observed. The results suggest a zinc hydroxide mechanism for Cab. The overall results indicate that prokaryotic β-class carbonic anhydrases have fundamental characteristics similar to the eukaryotic β-class enzymes and firmly establish that the α-, β-, and γ-classes are convergently evolved enzymes that, although structurally distinct, are functionally equivalent.
PMCID: PMC111400  PMID: 11073902
15.  A Plant-Type (β-Class) Carbonic Anhydrase in the Thermophilic Methanoarchaeon Methanobacterium thermoautotrophicum 
Journal of Bacteriology  1999;181(20):6247-6253.
Carbonic anhydrase, a zinc enzyme catalyzing the interconversion of carbon dioxide and bicarbonate, is nearly ubiquitous in the tissues of highly evolved eukaryotes. Here we report on the first known plant-type (β-class) carbonic anhydrase in the archaea. The Methanobacterium thermoautotrophicum ΔH cab gene was hyperexpressed in Escherichia coli, and the heterologously produced protein was purified 13-fold to apparent homogeneity. The enzyme, designated Cab, is thermostable at temperatures up to 75°C. No esterase activity was detected with p-phenylacetate as the substrate. The enzyme is an apparent tetramer containing approximately one zinc per subunit, as determined by plasma emission spectroscopy. Cab has a CO2 hydration activity with a kcat of 1.7 × 104 s−1 and Km for CO2 of 2.9 mM at pH 8.5 and 25°C. Western blot analysis indicates that Cab (β class) is expressed in M. thermoautotrophicum; moreover, a protein cross-reacting to antiserum raised against the γ carbonic anhydrase from Methanosarcina thermophila was detected. These results show that β-class carbonic anhydrases extend not only into the Archaea domain but also into the thermophilic prokaryotes.
PMCID: PMC103756  PMID: 10515911

Results 1-15 (15)