A Phosphofructokinase Homolog fromPyrobaculum calidifontisDisplays Kinase Activity towards Pyrimidine Nucleosides and Ribose 1-Phosphate

ABSTRACTThe genome of the hyperthermophilic archaeonPyrobaculum calidifontiscontains an open reading frame, Pcal_0041, annotated as encoding a PfkB family ribokinase, consisting of phosphofructokinase and pyrimidine kinase domains. Among the biochemically characterized enzymes, the Pcal_0041 protein was 37% identical to the phosphofructokinase (Ape_0012) fromAeropyrum pernix, which displayed kinase activity toward a broad spectrum of substrates, including sugars, sugar phosphates, and nucleosides, and 36% identical to a phosphofructokinase fromDesulfurococcus amylolyticus. To examine the biochemical function of the Pcal_0041 protein, we cloned and expressed the gene and purified the recombinant protein. Although the Pcal_0041 protein contained a putative phosphofructokinase domain, it exhibited only low levels of phosphofructokinase activity. The recombinant enzyme catalyzed the phosphorylation of nucleosides and, to a lower extent, sugars and sugar phosphates. Surprisingly, among the substrates tested, the highest activity was detected with ribose 1-phosphate (R1P), followed by cytidine and uridine. The catalytic efficiency (kcat/Km) toward R1P was 11.5 mM−1· s−1. ATP was the most preferred phosphate donor, followed by GTP. Activity measurements with cell extracts ofP. calidifontisindicated the presence of nucleoside phosphorylase activity, which would provide the means to generate R1P from nucleosides. The study suggests that, in addition to the recently identified ADP-dependent ribose 1-phosphate kinase (R1P kinase) inThermococcus kodakarensisthat functions in the pentose bisphosphate pathway, R1P kinase is also present in members of the Crenarchaeota.IMPORTANCEThe discovery of the pentose bisphosphate pathway inThermococcus kodakarensishas clarified how this archaeon can degrade nucleosides. Homologs of the enzymes of this pathway are present in many members of the Thermococcales, suggesting that this metabolism occurs in these organisms. However, this is not the case in other archaea, and degradation mechanisms for nucleosides or ribose 1-phosphate are still unknown. This study reveals an important first step in understanding nucleoside metabolism in Crenarchaeota and identifies an ATP-dependent ribose 1-phosphate kinase inPyrobaculum calidifontis. The enzyme is structurally distinct from previously characterized archaeal members of the ribokinase family and represents a group of proteins found in many crenarchaea.

Download Full-text

An Uncharacterized Member of the Ribokinase Family in Thermococcus kodakarensis Exhibits myo-Inositol Kinase Activity

Journal of Biological Chemistry ◽

10.1074/jbc.m113.457259 ◽

2013 ◽

Vol 288 (29) ◽

pp. 20856-20867 ◽

Cited By ~ 7

Author(s):

Takaaki Sato ◽

Masahiro Fujihashi ◽

Yukika Miyamoto ◽

Keiko Kuwata ◽

Eriko Kusaka ◽

...

Keyword(s):

Kinase Activity ◽

Three Dimensional ◽

Fold Increase ◽

Adenosine Kinase ◽

Dimensional Structure ◽

Uncharacterized Protein ◽

Thermococcus Kodakarensis ◽

Sugar Phosphates ◽

Biochemical Analyses ◽

Physiological Substrates

Here we performed structural and biochemical analyses on the TK2285 gene product, an uncharacterized protein annotated as a member of the ribokinase family, from the hyperthermophilic archaeon Thermococcus kodakarensis. The three-dimensional structure of the TK2285 protein resembled those of previously characterized members of the ribokinase family including ribokinase, adenosine kinase, and phosphofructokinase. Conserved residues characteristic of this protein family were located in a cleft of the TK2285 protein as in other members whose structures have been determined. We thus examined the kinase activity of the TK2285 protein toward various sugars recognized by well characterized ribokinase family members. Although activity with sugar phosphates and nucleosides was not detected, kinase activity was observed toward d-allose, d-lyxose, d-tagatose, d-talose, d-xylose, and d-xylulose. Kinetic analyses with the six sugar substrates revealed high Km values, suggesting that they were not the true physiological substrates. By examining activity toward amino sugars, sugar alcohols, and disaccharides, we found that the TK2285 protein exhibited prominent kinase activity toward myo-inositol. Kinetic analyses with myo-inositol revealed a greater kcat and much lower Km value than those obtained with the monosaccharides, resulting in over a 2,000-fold increase in kcat/Km values. TK2285 homologs are distributed among members of Thermococcales, and in most species, the gene is positioned close to a myo-inositol monophosphate synthase gene. Our results suggest the presence of a novel subfamily of the ribokinase family whose members are present in Archaea and recognize myo-inositol as a substrate.

Download Full-text

Identification and enzymatic analysis of an archaeal ATP-dependent serine kinase from the hyperthermophilic archaeon Staphylothermus marinus

Journal of Bacteriology ◽

10.1128/jb.00025-21 ◽

2021 ◽

Author(s):

Yasunobu Mori ◽

Hiroki Kawamura ◽

Takaaki Sato ◽

Takayuki Fujita ◽

Ryuhei Nagata ◽

...

Keyword(s):

Kinase Activity ◽

Sugar Metabolism ◽

Inorganic Polyphosphates ◽

Serine Kinase ◽

Hyperthermophilic Archaeon ◽

Thermococcus Kodakarensis ◽

Wide Range ◽

Genes Encoding ◽

Phosphate Donor ◽

Domains Of Life

Serine kinase catalyzes the phosphorylation of free serine (Ser) to produce O -phosphoserine (Sep). An ADP-dependent Ser kinase in the hyperthermophilic archaeon Thermococcus kodakarensis ( Tk -SerK) is involved in cysteine (Cys) biosynthesis and most likely Ser assimilation. An ATP-dependent Ser kinase in the mesophilic bacterium Staphylococcus aureus is involved in siderophore biosynthesis. Although proteins displaying various degrees of similarity with Tk -SerK are distributed in a wide range of organisms, it is unclear if they are actually Ser kinases. Here we examined proteins from Desulfurococcales species in Crenarchaeota that display moderate similarity with Tk -SerK from Euryarchaeota (42-45% identical). Tk - serK homologs from Staphylothermus marinus (Smar_0555), Desulfurococcus amylolyticus (DKAM_0858), and Desulfurococcus mucosus (Desmu_0904) were expressed in Escherichia coli . All three partially purified recombinant proteins exhibited Ser kinase activity utilizing ATP rather than ADP as a phosphate donor. Purified Smar_0555 protein displayed activity towards l -Ser, but not with other compounds including d -Ser, l -threonine and l -homoserine. The enzyme utilized ATP, UTP, GTP, CTP, and the inorganic polyphosphates triphosphate and tetraphosphate as the phosphate donor. Kinetic analysis indicated that the Smar_0555 protein preferred nucleoside 5’-triphosphates compared to triphosphate as a phosphate donor. Transcript levels and Ser kinase activity in S. marinus cells grown with or without serine suggested that the Smar_0555 gene is constitutively expressed. The genes encoding Ser kinases examined here form an operon with genes most likely responsible for the conversion between Sep and 3-phosphoglycerate of central sugar metabolism, suggesting that the ATP-dependent Ser kinases from Desulfurococcales play a role in the assimilation of Ser. IMPORTANCE Homologs of the ADP-dependent Ser kinase from the archaeon Thermococcus kodakarensis ( Tk -SerK) include representatives from all three domains of life. The results of this study show that even homologs from the archaeal order Desulfurococcales, which are the most structurally related to the ADP-dependent Ser kinases from the Thermococcales, are Ser kinases that utilize ATP, and in at least some cases inorganic polyphosphates, as the phosphate donor. The differences in properties between the Desulfurococcales and Thermococcales enzymes raise the possibility that Tk -SerK homologs constitute a group of kinases that phosphorylate free serine with a wide range of phosphate donors.

Download Full-text

Isolation of a Gene Responsible for the Oxidation oftrans-Anethole topara-Anisaldehyde by Pseudomonas putida JYR-1 and Its Expression in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.00781-12 ◽

2012 ◽

Vol 78 (15) ◽

pp. 5238-5246 ◽

Cited By ~ 9

Author(s):

Dongfei Han ◽

Ji-Young Ryu ◽

Robert A. Kanaly ◽

Hor-Gil Hur

Keyword(s):

Escherichia Coli ◽

Pseudomonas Putida ◽

Hypothetical Protein ◽

Aldehyde Group ◽

Agrobacterium Vitis ◽

T7 Promoter ◽

Content Type ◽

E Coli ◽

Cell Extracts ◽

Isoeugenol Monooxygenase

ABSTRACTA plasmid, pTA163, inEscherichia colicontained an approximately 34-kb gene fragment fromPseudomonas putidaJYR-1 that included the genes responsible for the metabolism oftrans-anethole to protocatechuic acid. Three Tn5-disrupted open reading frame 10 (ORF 10) mutants of plasmid pTA163 lost their abilities to catalyzetrans-anethole. Heterologously expressed ORF 10 (1,047 nucleotides [nt]) under a T7 promoter inE. colicatalyzed oxidative cleavage of a propenyl group oftrans-anethole to an aldehyde group, resulting in the production ofpara-anisaldehyde, and this gene was designatedtao(trans-anetholeoxygenase). The deduced amino acid sequence of TAO had the highest identity (34%) to a hypothetical protein ofAgrobacterium vitisS4 and likely contained a flavin-binding site. Preferred incorporation of an oxygen molecule from water intop-anisaldehyde using18O-labeling experiments indicated stereo preference of TAO for hydrolysis of the epoxide group. Interestingly, unlike the narrow substrate range of isoeugenol monooxygenase fromPseudomonas putidaIE27 andPseudomonas nitroreducensJin1, TAO fromP. putidaJYR-1 catalyzed isoeugenol,O-methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure. Addition of NAD(P)H to the ultrafiltered cell extracts ofE. coli(pTA163) increased the activity of TAO. Due to the relaxed substrate range of TAO, it may be utilized for the production of various fragrance compounds from plant phenylpropanoids in the future.

Download Full-text

Topology of Streptococcus pneumoniae CpsC, a Polysaccharide Copolymerase and Bacterial Protein Tyrosine Kinase Adaptor Protein

Journal of Bacteriology ◽

10.1128/jb.02106-14 ◽

2014 ◽

Vol 197 (1) ◽

pp. 120-127 ◽

Cited By ~ 4

Author(s):

Jonathan J. Whittall ◽

Renato Morona ◽

Alistair J. Standish

Keyword(s):

Streptococcus Pneumoniae ◽

Tyrosine Kinase ◽

Tyrosine Kinases ◽

Kinase Activity ◽

Tyrosine Phosphatase ◽

Adaptor Protein ◽

Tyrosine Kinase Activity ◽

Content Type ◽

Protein Tyrosine ◽

C Terminus

In Gram-positive bacteria, tyrosine kinases are split into two proteins, the cytoplasmic tyrosine kinase and a transmembrane adaptor protein. InStreptococcus pneumoniae, this transmembrane adaptor is CpsC, with the C terminus of CpsC critical for interaction and subsequent tyrosine kinase activity of CpsD. Topology predictions suggest that CpsC has two transmembrane domains, with the N and C termini present in the cytoplasm. In order to investigate CpsC topology, we used a chromosomal hemagglutinin (HA)-tagged Cps2C protein inS. pneumoniaestrain D39. Incubation of both protoplasts and membranes with carboxypeptidase B (CP-B) resulted in complete degradation of HA-Cps2C in all cases, indicating that the C terminus of Cps2C was likely extracytoplasmic and hence that the protein's topology was not as predicted. Similar results were seen with membranes fromS. pneumoniaestrain TIGR4, indicating that Cps4C also showed similar topology. A chromosomally encoded fusion of HA-Cps2C and Cps2D was not degraded by CP-B, suggesting that the fusion fixed the C terminus within the cytoplasm. However, capsule synthesis was unaltered by this fusion. Detection of the CpsC C terminus by flow cytometry indicated that it was extracytoplasmic in approximately 30% of cells. Interestingly, a mutant in the protein tyrosine phosphatase CpsB had a significantly greater proportion of positive cells, although this effect was independent of its phosphatase activity. Our data indicate that CpsC possesses a varied topology, with the C terminus flipping across the cytoplasmic membrane, where it interacts with CpsD in order to regulate tyrosine kinase activity.

Download Full-text

Analysis of tyrosine kinase activity in cell extracts using nondenaturing polyacrylamide gel electrophoresis

Analytical Biochemistry ◽

10.1016/0003-2697(87)90388-5 ◽

1987 ◽

Vol 164 (1) ◽

pp. 214-220 ◽

Cited By ~ 17

Author(s):

Robert I. Glazer ◽

Gang Yu ◽

Marian C. Knode

Keyword(s):

Tyrosine Kinase ◽

Gel Electrophoresis ◽

Kinase Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Polyacrylamide Gel ◽

Tyrosine Kinase Activity ◽

Cell Extracts

Download Full-text

Comparative Profiling and Discovery of Novel Glycosylated Mycosporine-Like Amino Acids in Two Strains of the Cyanobacterium Scytonema cf. crispum

Applied and Environmental Microbiology ◽

10.1128/aem.01633-16 ◽

2016 ◽

Vol 82 (19) ◽

pp. 5951-5959 ◽

Cited By ~ 19

Author(s):

Paul M. D'Agostino ◽

Vivek S. Javalkote ◽

Rabia Mazmouz ◽

Russell Pickford ◽

Pravin R. Puranik ◽

...

Keyword(s):

Amino Acids ◽

New Zealand ◽

Uv Radiation ◽

High Performance ◽

Parent Compound ◽

Future Research ◽

Content Type ◽

Chromatography Analysis ◽

Paralytic Shellfish Toxins ◽

Cell Extracts

ABSTRACTThe mycosporine-like amino acids (MAAs) are a group of small molecules with a diverse ecological distribution among microorganisms. MAAs have a range of physiological functions, including protection against UV radiation, making them important from a biotechnological perspective. In the present study, we identified a putative MAA (mys) gene cluster in two New Zealand isolates ofScytonemacf.crispum(UCFS10 and UCFS15). Homology to “Anabaena-type”mysclusters suggested that this cluster was likely to be involved in shinorine biosynthesis. Surprisingly, high-performance liquid chromatography analysis ofS. cf.crispumcell extracts revealed a complex MAA profile, including shinorine, palythine-serine, and their hexose-bound variants. It was hypothesized that a short-chain dehydrogenase (UCFS15_00405) encoded by a gene adjacent to theS. cf.crispummyscluster was responsible for the conversion of shinorine to palythine-serine. Heterologous expression of MysABCE and UCFS15_00405 inEscherichia coliresulted in the exclusive production of the parent compound shinorine. Taken together, these results suggest that shinorine biosynthesis inS. cf.crispumproceeds via anAnabaena-type mechanism and that the genes responsible for the production of other MAA analogues, including palythine-serine and glycosylated analogues, may be located elsewhere in the genome.IMPORTANCERecently, New Zealand isolates ofS. cf.crispumwere linked to the production of paralytic shellfish toxins for the first time, but no other natural products from this species have been reported. Thus, the species was screened for important natural product biosynthesis. The mycosporine-like amino acids (MAAs) are among the strongest absorbers of UV radiation produced in nature. The identification of novel MAAs is important from a biotechnology perspective, as these molecules are able to be utilized as sunscreens. This study has identified two novel MAAs that have provided several new avenues of future research related to MAA genetics and biosynthesis. Further, we have revealed that the genetic basis of MAA biosynthesis may not be clustered on the genome. The identification of the genes responsible for MAA biosynthesis is vital for future genetic engineering.

Download Full-text

Single-cell sphingosine kinase activity measurements in primary leukemia

Analytical and Bioanalytical Chemistry ◽

10.1007/s00216-014-7974-6 ◽

2014 ◽

Vol 406 (27) ◽

pp. 7027-7036 ◽

Cited By ~ 11

Author(s):

Alexandra J. Dickinson ◽

Sally A. Hunsucker ◽

Paul M. Armistead ◽

Nancy L. Allbritton

Keyword(s):

Single Cell ◽

Kinase Activity ◽

Sphingosine Kinase ◽

Activity Measurements ◽

Primary Leukemia

Download Full-text

Causal Role of Single Nucleotide Polymorphisms within themprFGene of Staphylococcus aureus in Daptomycin Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01184-13 ◽

2013 ◽

Vol 57 (11) ◽

pp. 5658-5664 ◽

Cited By ~ 48

Author(s):

Soo-Jin Yang ◽

Nagendra N. Mishra ◽

Aileen Rubio ◽

Arnold S. Bayer

Keyword(s):

Staphylococcus Aureus ◽

Single Nucleotide Polymorphisms ◽

Point Mutations ◽

Nucleotide Polymorphisms ◽

Wild Type ◽

Single Nucleotide ◽

Content Type ◽

Reading Frame ◽

A Charge

ABSTRACTSingle nucleotide polymorphisms (SNPs) within themprFopen reading frame (ORF) have been commonly observed in daptomycin-resistant (DAPr)Staphylococcus aureusstrains. Such SNPs are usually associated with a gain-in-function phenotype, in terms of either increased synthesis or enhanced translocation (flipping) of lysyl-phosphatidylglycerol (L-PG). However, it is unclear if suchmprFSNPs are causal in DAPrstrains or are merely a biomarker for this phenotype. In this study, we used an isogenic set ofS. aureusstrains: (i) Newman, (ii) its isogenic ΔmprFmutant, and (iii) several intransplasmid complementation constructs, expressing either a wild-type or point-mutated form of themprFORF cloned from two isogenic DAP-susceptible (DAPs)-DAPrstrain pairs (616-701 and MRSA11/11-REF2145). Complementation of the ΔmprFstrain with singly point-mutatedmprFgenes (mprFS295LormprFT345A) revealed that (i) individual and distinct point mutations within themprFORF can recapitulate phenotypes observed in donor strains (i.e., changes in DAP MICs, positive surface charge, and cell membrane phospholipid profiles) and (ii) these gain-in-function SNPs (i.e., enhanced L-PG synthesis) likely promote reduced DAP binding toS. aureusby a charge repulsion mechanism. Thus, for these two DAPrstrains, the definedmprFSNPs appear to be causally related to this phenotype.

Download Full-text

Application of a Euryarchaeota-Specific Helicase from Thermococcus kodakarensis for Noise Reduction in PCR

Applied and Environmental Microbiology ◽

10.1128/aem.04116-15 ◽

2016 ◽

Vol 82 (10) ◽

pp. 3022-3031 ◽

Cited By ~ 8

Author(s):

Ayako Fujiwara ◽

Katsuhiro Kawato ◽

Saori Kato ◽

Kiyoshi Yasukawa ◽

Ryota Hidese ◽

...

Keyword(s):

Noise Reduction ◽

Dna Polymerase ◽

Biological Activities ◽

Genetic Diagnosis ◽

Gc Content ◽

Dna Amplification ◽

16S Rrna Genes ◽

Rrna Genes ◽

Content Type ◽

Thermococcus Kodakarensis

ABSTRACTDNA/RNA helicases, which are enzymes for eliminating hydrogen bonds between bases of DNA/DNA, DNA/RNA, and RNA/RNA using the energy of ATP hydrolysis, contribute to various biological activities. In the present study, theEuryarchaeota-specific helicase EshA (TK0566) from the hyperthermophilic archaeonThermococcus kodakarensis(Tk-EshA) was obtained as a recombinant form, and its enzymatic properties were examined.Tk-EshA exhibited maximal ATPase activity in the presence of RNA at 80°C. Unwinding activity was evaluated with various double-stranded DNAs (forked, 5′ overhung, 3′ overhung, and blunt end) at 50°C.Tk-EshA unwound forked and 3′ overhung DNAs. These activities were expected to unwind the structured template and to peel off misannealed primers whenTk-EshA was added to a PCR mixture. To examine the effect ofTk-EshA on PCR, various target DNAs were selected, and DNA synthesis was investigated. When 16S rRNA genes were used as a template, several misamplified products (noise DNAs) were detected in the absence ofTk-EshA. In contrast, noise DNAs were eliminated in the presence ofTk-EshA. Noise reduction byTk-EshA was also confirmed whenTaqDNA polymerase (a family A DNA polymerase, PolI type) and KOD DNA polymerase (a family B DNA polymerase, α type) were used for PCR. Misamplified bands were also eliminated duringtoxAgene amplification fromPseudomonas aeruginosaDNA, which possesses a high GC content (69%).Tk-EshA addition was more effective than increasing the annealing temperature to reduce misamplified DNAs duringtoxAamplification.Tk-EshA is a useful tool to reduce noise DNAs for accurate PCR.IMPORTANCEPCR is a technique that is useful for genetic diagnosis, genetic engineering, and detection of pathogenic microorganisms. However, troubles with nonspecific DNA amplification often occur from primer misannealing. In order to achieve a specific DNA amplification by eliminating noise DNAs derived from primer misannealing, a thermostableEuryarchaeota-specific helicase (Tk-EshA) was included in the PCR mixture. The addition ofTk-EshA has reduced noise DNAs in PCR.

Download Full-text