Expression of Functional Streptomyces coelicolor H+-Pyrophosphatase and Characterization of Its Molecular Properties

Hirono, Megumi; Mimura, Hisatoshi; Nakanishi, Yoichi; Maeshima, Masayoshi

doi:10.1093/jb/mvi112

Cited by 15 publications

(10 citation statements)

References 39 publications

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“…This structural feature matches very well with the phylogenetic clustering of protein sequences into the two groups mentioned above. However, the opposed change (from K to A) does not appear to provide K þ -dependency to a K þ -insensitive H þ -PPase, as shown with the Streptomyces coelicolor protein (39). Thus, other structural determinants seem to be needed to confer this functional feature, as was also suggested by sequence comparison studies (35 -37).…”

Section: The Identification Of Functionally Important Conserved Motifmentioning

confidence: 86%

H+‐PPases: yesterday, today and tomorrow

et al. 2007

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SummarySuggestions by Calvin about a role of inorganic pyrophosphate (PPi) in early photosynthesis and by Lipmann that PPi may have been the original energy-rich phosphate donor in biological energy conversion, were followed in the mid-1960s by experimental results with isolated chromatophore membranes from the purple photosynthetic bacterium Rhodospirillum rubrum. PPi was shown to be hydrolysed in an uncoupler stimulated reaction by a membranebound inorganic pyrophosphatase (PPase), to be formed at the expense of light energy in photophosphorylation and to be utilized as an energy donor for various energy-requiring reactions, as a first known alternative to ATP. This direct link between PPi and photosynthesis led to increasing attention concerning the role of PPi in both early and present biological energy transfer. In the 1970s, the PPase was shown to be a proton pump and to be present also in higher plants. In the 1990s, sequences of H þ -PPase genes were obtained from plants, protists, bacteria and archaea and two classes of H þ -PPases differing in K þ sensitivity were established. Over 200 H þ -PPase sequences have now been determined. Recent biochemical and biophysical results have led to new progress and questions regarding the H þ -PPase family, as well as the families of soluble PPases and the inorganic polyphosphatases, which hydrolyse inorganic linear high-molecular-weight polyphosphates (HMWpolyP). Here we will focus attention on the H þ -PPases, their evolution and putative active site motifs, response to monovalent cations, genetic regulation and some very recent results, based on new methods for obtaining large quantities of purified protein, about their tertiary and quaternary structures. IUBMB Life, 59: 76-83, 2007

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Section: The Identification Of Functionally Important Conserved Motifmentioning

confidence: 86%

H+‐PPases: yesterday, today and tomorrow

et al. 2007

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“…The gene for cp157-Venus was generated by circular permutation from yEVenus of pKT90 (EUROSCARF) optimized for yeast expression. One MoBD was amplified from E.coli genomic DNA by PCR, another was originally designed by Gene-Synthesizer software [25], [26] and artificially generated from synthetic oligo-DNAs by ligase-chain reaction. Both MoBDs correspond to c-terminal halves of E.coli ModE.…”

Section: Methodsmentioning

confidence: 99%

Exploring Dynamics of Molybdate in Living Animal Cells by a Genetically Encoded FRET Nanosensor

et al. 2013

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Molybdenum (Mo) is an essential trace element for almost all living organisms including animals. Mo is used as a catalytic center of molybdo-enzymes for oxidation/reduction reactions of carbon, nitrogen, and sulfur metabolism. Whilst living cells are known to import inorganic molybdate oxyanion from the surrounding environment, the in vivo dynamics of cytosolic molybdate remain poorly understood as no appropriate indicator is available for this trace anion. We here describe a genetically encoded Förester-resonance-energy-transfer (FRET)-based nanosensor composed of CFP, YFP and the bacterial molybdate-sensor protein ModE. The nanosensor MolyProbe containing an optimized peptide-linker responded to nanomolar-range molybdate selectively, and increased YFP:CFP fluorescence intensity ratio by up to 109%. By introduction of the nanosensor, we have been able to successfully demonstrate the real-time dynamics of molybdate in living animal cells. Furthermore, time course analyses of the dynamics suggest that novel oxalate-sensitive- and sulfate-resistant- transporter(s) uptake molybdate in a model culture cell.

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“…GNTTAA is considered to be an important H + -PPase domain because it could be related to potassium- dependence, especially due to the first A site [6], [29]. This site has two possible forms: A and K. A is a K + -dependent native and K is a K + -independent variant [6], [29]. In each of the 14 novel H + -PPases from 7 eremophytes, domain 3 exists as GNTTAA (Figure 1) and is located between the 10th and the 11th transmembrane region of SaVP1 (Figure 2).…”

Section: Resultsmentioning

confidence: 99%

“…The GNTTAA domain is considered to be a marker of type I/II, according to the first A site [6]. This site has two possible forms, A and K. It was believed that the A form is the K + - dependent H + -PPase and the K form is K + -independent H + -PPase [6], [29], [37]. This domain is GNTTAA in all 14 novel eremophyte H + -PPases.…”

Section: Discussionmentioning

confidence: 99%

Isolation and Characterization of a Conserved Domain in the Eremophyte H+-PPase Family

et al. 2013

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H+-translocating inorganic pyrophosphatases (H+-PPase) were recognized as the original energy donors in the development of plants. A large number of researchers have shown that H+-PPase could be an early-originated protein that participated in many important biochemical and physiological processes. In this study we cloned 14 novel sequences from 7 eremophytes: Sophora alopecuroid (Sa), Glycyrrhiza uralensis (Gu), Glycyrrhiza inflata (Gi), Suaeda salsa (Ss), Suaeda rigida (Sr), Halostachys caspica (Hc), and Karelinia caspia (Kc). These novel sequences included 6 ORFs and 8 fragments, and they were identified as H+-PPases based on the typical conserved domains. Besides the identified domains, sequence alignment showed that there still were two novel conserved motifs. A phylogenetic tree was constructed, including the 14 novel H+-PPase amino acid sequences and the other 34 identified H+-PPase protein sequences representing plants, algae, protozoans and bacteria. It was shown that these 48 H+-PPases were classified into two groups: type I and type II H+-PPase. The novel 14 eremophyte H+-PPases were classified into the type I H+-PPase. The 3D structures of these H+-PPase proteins were predicted, which suggested that all type I H+-PPases from higher plants and algae were homodimers, while other type I H+-PPases from bacteria and protozoans and all type II H+-PPases were monomers. The 3D structures of these novel H+-PPases were homodimers except for SaVP3, which was a monomer. This regular structure could provide important evidence for the evolutionary origin and study of the relationship between the structure and function among members of the H+-PPase family.

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Expression of Functional Streptomyces coelicolor H+-Pyrophosphatase and Characterization of Its Molecular Properties

Cited by 15 publications

References 39 publications

H+‐PPases: yesterday, today and tomorrow

H+‐PPases: yesterday, today and tomorrow

Exploring Dynamics of Molybdate in Living Animal Cells by a Genetically Encoded FRET Nanosensor

Isolation and Characterization of a Conserved Domain in the Eremophyte H+-PPase Family

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