Different Physiological Roles of ATP- and PP<sub>i</sub>-Dependent Phosphofructokinase Isoenzymes in the Methylotrophic Actinomycete<i>Amycolatopsis methanolica</i>

Alves, Alexandra Möller; Euverink, Gert-Jan; Santos, Helena; Dijkhuizen, Lubbert

doi:10.1128/jb.183.24.7231-7240.2001

Cited by 51 publications

(30 citation statements)

References 43 publications

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“…Six pBM19 genes, together with two chromosomal genes, are transcriptionally induced upon growth of B. methanolicus in methanol. It has been shown that expression of genes may be selectively induced upon C 1 growth in autotrophic (27) and methylotrophic (1,3,15,17,29) bacteria. To fully explore the biological function of pBM19 for methylotrophic growth, we analyzed the transcriptional regulation of a selection of its genes by RT-PCR.…”

Section: Resultsmentioning

confidence: 99%

Upregulated Transcription of Plasmid and Chromosomal Ribulose Monophosphate Pathway Genes Is Critical for Methanol Assimilation Rate and Methanol Tolerance in the Methylotrophic Bacterium Bacillus methanolicus

et al. 2006

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The natural plasmid pBM19 carries the key mdh gene needed for the oxidation of methanol into formaldehyde by Bacillus methanolicus. Five more genes, glpX, fba, tkt, pfk, and rpe, with deduced roles in the cell primary metabolism, are also located on this plasmid. By using real-time PCR, we show that they are transcriptionally upregulated (6-to 40-fold) in cells utilizing methanol; a similar induction was shown for two chromosomal genes, hps and phi. These seven genes are involved in the fructose bisphosphate aldolase/sedoheptulose bisphosphatase variant of the ribulose monophosphate (RuMP) pathway for formaldehyde assimilation. Curing of pBM19 causes higher methanol tolerance and reduced formaldehyde tolerance, and the methanol tolerance is reversed to wild-type levels by reintroducing mdh. Thus, the RuMP pathway is needed to detoxify the formaldehyde produced by the methanol dehydrogenase-mediated conversion of methanol, and the in vivo transcription levels of mdh and the RuMP pathway genes reflect the methanol tolerance level of the cells. The transcriptional inducer of hps and phi genes is formaldehyde, and not methanol, and introduction of multiple copies of these two genes into B. methanolicus made the cells more tolerant of growth on high methanol concentrations. The recombinant strain also had a significantly higher specific growth rate on methanol than the wild type. While pBM19 is critical for growth on methanol and important for formaldehyde detoxification, the maintenance of this plasmid represents a burden for B. methanolicus when growing on mannitol. Our data contribute to a new and fundamental understanding of the regulation of B. methanolicus methylotrophy.Aerobic methylotrophs are bacteria capable of utilizing reduced one-carbon (C 1 ) compounds as the sole carbon source for growth and energy (2), and the majority of research on these bacteria has focused on their biochemical novelty and commercial viability. A number of gram-positive and thermotolerant Bacillus strains have been isolated and designated Bacillus methanolicus (4,26). This methylotrophic bacterium has a novel NAD-dependent methanol dehydrogenase (MDH), which contains bound NAD, to oxidize methanol into formaldehyde (11). The enzyme has a remarkably high affinity for methanol, and an activator protein called ACT modulates its in vivo activity. ACT activates MDH by hydrolysis, and the expression of these two proteins is reported to be under coordinate (and methanol-induced) control in B. methanolicus (16). Formaldehyde is the key intermediate in C 1 metabolism and can be assimilated via the ribulose monophosphate (RuMP) pathway (Fig. 1). Many RuMP pathway variants are described in the literature, and thermotolerant Bacillus strains are reported to use the fructosebisphosphate aldolase/transaldolase variant (3,4,12).Steady-state cultures of B. methanolicus MGA3 limited by methanol in the feed display sensitivity to minor methanol pulses and respond by a transient decline in biomass concentration. By using 13 C nuclear magnetic resonan...

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Section: Resultsmentioning

confidence: 99%

Upregulated Transcription of Plasmid and Chromosomal Ribulose Monophosphate Pathway Genes Is Critical for Methanol Assimilation Rate and Methanol Tolerance in the Methylotrophic Bacterium Bacillus methanolicus

et al. 2006

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“…Examples of organisms that express both PP i -PFK and ATP-PFK are rare. The actinomycete Amycolatopsis methanolica possesses both genes, but their expression depends strictly on the carbon source (46). Entamoeba histolytica possesses two genes for PP i -PFK orthologues; however, one of the gene products has been shown to utilize ATP instead of PP i , and it has been suggested that the two enzymes might be expressed during different life stages (37).…”

Section: Figmentioning

confidence: 99%

N-Terminal Presequence-Independent Import of Phosphofructokinase into Hydrogenosomes of Trichomonas vaginalis

et al. 2015

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bMitochondrial evolution entailed the origin of protein import machinery that allows nuclear-encoded proteins to be targeted to the organelle, as well as the origin of cleavable N-terminal targeting sequences (NTS) that allow efficient sorting and import of matrix proteins. In hydrogenosomes and mitosomes, reduced forms of mitochondria with reduced proteomes, NTS-independent targeting of matrix proteins is known. Here, we studied the cellular localization of two glycolytic enzymes in the anaerobic pathogen Trichomonas vaginalis: PP i -dependent phosphofructokinase (TvPP i -PFK), which is the main glycolytic PFK activity of the protist, and ATP-dependent PFK (TvATP-PFK), the function of which is less clear. TvPP i -PFK was detected predominantly in the cytosol, as expected, while all four TvATP-PFK paralogues were imported into T. vaginalis hydrogenosomes, although none of them possesses an NTS. The heterologous expression of TvATP-PFK in Saccharomyces cerevisiae revealed an intrinsic capability of the protein to be recognized and imported into yeast mitochondria, whereas yeast ATP-PFK resides in the cytosol. TvATP-PFK consists of only a catalytic domain, similarly to "short" bacterial enzymes, while ScATP-PFK includes an N-terminal extension, a catalytic domain, and a C-terminal regulatory domain. Expression of the catalytic domain of ScATP-PFK and short Escherichia coli ATP-PFK in T. vaginalis resulted in their partial delivery to hydrogenosomes. These results indicate that TvATP-PFK and the homologous ATP-PFKs possess internal structural targeting information that is recognized by the hydrogenosomal import machinery. From an evolutionary perspective, the predisposition of ancient ATP-PFK to be recognized and imported into hydrogenosomes might be a relict from the early phases of organelle evolution.T he transition of the mitochondrion into an ATP-producing organelle was the crucial event at the eukaryote origin (1). ATP synthesis in eukaryotes is typically compartmentalized, with glycolysis in the cytosol and pyruvate oxidation in the mitochondria, which is linked to highly efficient oxidative phosphorylation (1, 2). In protists, however, there are notable exceptions to the usual scheme regarding both glycolysis and pyruvate oxidation. In Trichomonas vaginalis and other eukaryotes that possess an anaerobic form of mitochonria called hydrogenosomes, pyruvate is oxidized within the organelle via less efficient anaerobic fermentation (3). Giardia intestinalis, Entamoeba histolytica, and other eukaryotes possess a reduced form of mitochondria called mitosomes that do not produce ATP at all (4). In these organisms, pyruvate oxidation takes place exclusively in the cytosol (1). In kinetoplastids, glycolysis is compartmentalized in specialized microbodies called glycosomes (5). In some green algae, the first half of the glycolytic pathway is localized in the chloroplast (6, 7), while in the diatom Phaeodactylum tricornutum and other stramenopiles, several glycolytic enzymes are targeted to multiple compartments, such...

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“…However, an ATP-Pfk is induced during cultivation on one-carbon compounds, e.g. methanol (53). Based on the unique PpPfk structure suggested above, one can speculate that the existence of a particular Pfk species may be of advantage for methanol-assimilating yeasts such as Pichia.…”

Section: Discussionmentioning

confidence: 98%

A Novel Form of 6-Phosphofructokinase

Tanneberger

Kirchberger

Bär

et al. 2007

Journal of Biological Chemistry

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Classically, 6-phosphofructokinases are homo-and heterooligomeric enzymes consisting of ␣ subunits and ␣/␤ subunits, respectively. Herein, we describe a new form of 6-phosphofructokinase (Pfk) present in several Pichia species, which is composed of three different types of subunit, ␣, ␤, and ␥. The sequence of the ␥ subunit shows no similarity to classic Pfk subunits or to other known protein sequences. In-depth structural and functional studies revealed that the ␥ subunit is a constitutive component of Pfk from Pichia pastoris (PpPfk). Analyses of the purified PpPfk suggest a heterododecameric assembly from the three different subunits. Accordingly, it is the largest and most complex Pfk identified yet. Although, the ␥ subunit is not required for enzymatic activity, the ␥ subunit-deficient mutant displays a decreased growth on nutrient limitation and reduced cell flocculation when compared with the P. pastoris wild-type strain. Subsequent characterization of purified Pfks from wildtype and ␥ subunit-deficient strains revealed that the allosteric regulation of the PpPfk by ATP, fructose 2,6-bisphosphate, and AMP is fine-tuned by the ␥ subunit. Therefore, we suggest that the ␥ subunit contributes to adaptation of P. pastoris to energy resources.The ATP-dependent 6-phosphofructokinase (EC 2.7.1.11, phosphofructokinase-1, ATP:D-fructose-6-phosphate 1-phosphotransferase (Pfk)) 2 catalyzes in many organisms the phosphorylation of fructose 6-phosphate (Fru 6-P) at position 1. The Pfk activity is generally being sensitive to a number of allosteric regulators, e.g. ATP, AMP, NH 4 ϩ , and fructose 2,6-bisphosphate (Fru 2,6-P 2 ). Therefore, this irreversible reaction is considered to be one of the rate-limiting steps of glycolysis (1-3). Most eukaryotic Pfks are heteromeric enzymes consisting of subunits, which evolved from a single ancestor gene by gene duplication and mutational events (4, 5). Specific amino acid residues involved in catalytic and regulatory functions of Pfk from Escherichia coli (6, 7) are conserved in yeast and mammalian Pfk genes. In eukaryotes the N-terminal half of a Pfk subunit obviously retained the catalytic function, whereas in the C-terminal half allosteric ligand binding sites have evolved from former catalytic and regulatory sites (4,8,9). This assumption is supported by studies with mutants of Saccharomyces cerevisiae expressing only the ␣ or the ␤ subunit of Pfk. It was demonstrated that one subunit type alone is able to form an enzymatically active Pfk entity in vivo (10, 11). Crystallographic analysis showed that an active bacterial Pfk consists of four identical subunits (12, 13). No high resolution structure of a eukaryotic Pfk is available yet. But electron microscopic studies with S. cerevisiae Pfk (ScPfk) at 10.8-Å resolution suggested an octameric enzyme assembly (14).Recently we co-purified a protein component together with the known Pfk ␣ and ␤ subunits (15-17) from the methylotrophic yeast Pichia pastoris. This unknown protein could only be separated under denaturating conditio...

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Different Physiological Roles of ATP- and PP_i-Dependent Phosphofructokinase Isoenzymes in the Methylotrophic ActinomyceteAmycolatopsis methanolica

Cited by 51 publications

References 43 publications

Upregulated Transcription of Plasmid and Chromosomal Ribulose Monophosphate Pathway Genes Is Critical for Methanol Assimilation Rate and Methanol Tolerance in the Methylotrophic Bacterium Bacillus methanolicus

Upregulated Transcription of Plasmid and Chromosomal Ribulose Monophosphate Pathway Genes Is Critical for Methanol Assimilation Rate and Methanol Tolerance in the Methylotrophic Bacterium Bacillus methanolicus

N-Terminal Presequence-Independent Import of Phosphofructokinase into Hydrogenosomes of Trichomonas vaginalis

A Novel Form of 6-Phosphofructokinase

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Different Physiological Roles of ATP- and PPi-Dependent Phosphofructokinase Isoenzymes in the Methylotrophic ActinomyceteAmycolatopsis methanolica

Cited by 51 publications

References 43 publications

Upregulated Transcription of Plasmid and Chromosomal Ribulose Monophosphate Pathway Genes Is Critical for Methanol Assimilation Rate and Methanol Tolerance in the Methylotrophic Bacterium Bacillus methanolicus

Upregulated Transcription of Plasmid and Chromosomal Ribulose Monophosphate Pathway Genes Is Critical for Methanol Assimilation Rate and Methanol Tolerance in the Methylotrophic Bacterium Bacillus methanolicus

N-Terminal Presequence-Independent Import of Phosphofructokinase into Hydrogenosomes of Trichomonas vaginalis

A Novel Form of 6-Phosphofructokinase

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Different Physiological Roles of ATP- and PP_i-Dependent Phosphofructokinase Isoenzymes in the Methylotrophic ActinomyceteAmycolatopsis methanolica