Crystal structure of Escherichia coli inorganic pyrophosphatase complexed with SO42−

Avaeva, S.M.; Kurilova, S. A.; Nazarova, T. I.; Rodina, E. V.; Vorobyeva, N. N.; Sklyankina, V. A.; Grigorjeva, Olga; Harutyunyan, E.H.; Oganessyan, Vaheh; Wilson, K.S.; Dauter, Z.; Huber, Robert; Mather, Timothy

doi:10.1016/s0014-5793(97)00650-9

Cited by 30 publications

(24 citation statements)

References 12 publications

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“…Three differences between the two enzymes are apparent. First, the curve for mtPPase was markedly shifted to lower pH values, consistent with the proposed role for Asp 26 in ecPPase. Second, the final activity level reached in the acidic medium was lower for mtPPase.…”

Section: Catalytic Characteristics Of Magnesium-activated Mtppase Andsupporting

confidence: 77%

“…His 86 forms a hydrogen bond with a single bound sulfate ion, which mimics the product phosphate and occupies the same position as the sulfate in the ecPPase-sulfate complex (PDB code 1JFD; Ref. 26). The sulfate in mtPPase has an occupancy of 1, whereas the ecPPase sulfate has an occupancy of only 0.5, which may indicate stronger binding in mtPPase because of the bond with His 86 .…”

Section: Resultsmentioning

confidence: 99%

“…Formation of this bond causes a shift of up to 3.9 Å in the main chain segment 85-91. Another difference between the two enzymes is that sulfate binding to mtPPase does not cause the molecular asymmetry observed in ecPPase (26); the biological hexamer is formed by identical subunits in the sulfate complex of mtPPase.…”

Section: Resultsmentioning

confidence: 99%

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An Unusual, His-dependent Family I Pyrophosphatase from Mycobacterium tuberculosis

Tammenkoski

Benini

Magretova

et al. 2005

Journal of Biological Chemistry

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Soluble inorganic pyrophosphatases (PPases) comprise two evolutionarily unrelated families (I and II). These two families have different specificities for metal cofactors, which is thought to be because of the fact that family II PPases have three active site histidines, whereas family I PPases have none. Here, we report the structural and functional characterization of a unique family I PPase from Mycobacterium tuberculosis (mtPPase) that has two His residues (His 21 and His 86 ) in the active site. The 1.3-Å three-dimensional structure of mtPPase shows that His 86 directly interacts with bound sulfate, which mimics the product phosphate. Otherwise, mtPPase is structurally very similar to the well studied family I hexameric PPase from Escherichia coli, although mtPPase lacks the intersubunit metal binding site found in E. coli PPase. The cofactor specificity of mtPPase resembles that of E. coli PPase in that it has high activity in the presence of Mg 2؉ , but it differs from the E. coli enzyme and family II PPases because it has much lower activity in the presence of Mn 2؉ or Zn 2؉ . Replacements of His 21 and His 86 in mtPPase with the residues found in the corresponding positions of E. coli PPase had either no effect on the Mg 2؉ -and Mn 2؉ -supported reactions (H86K) or reduced Mg 2؉ -supported activity (H21K). However, both replacements markedly increased the Zn 2؉ -supported activity of mtPPase (up to 11-fold). In the double mutant, Zn 2؉ was a 2.5-fold better cofactor than Mg 2؉ . These results show that the His residues in mtPPase are not essential for catalysis, although they determine cofactor specificity. Soluble inorganic pyrophosphatase (PPase)3 (EC 3.6.1.1), is an essential metal-dependent enzyme that converts pyrophosphate into orthophosphate. This simple reaction provides a thermodynamic pull for many biosynthetic reactions that yield pyrophosphate as a byproduct (1). Soluble PPases belong to two nonhomologous families (2, 3): family I PPases, which are fairly widespread in all types of organisms, and family II PPases, which are exclusive to bacteria. In eubacteria and archaebacteria, the family I PPases are usually homohexamers, whereas in eukaryotes, they are homodimers. In contrast, all family II PPases are homodimers. The subunit size is generally 19 -22 kDa in hexameric PPases and 31-34 kDa in dimeric forms. Despite the variability of the subunit size in family I PPases, their active site cavities are formed by the same 13 functionally important polar residues, which is reflected in the conservation of the catalytic mechanism (4, 5). Although no sequence or overall structural similarity is observed between the two families, there is a striking similarity in the spatial arrangement of six key active site residues, a remarkable example of convergent enzyme evolution (6, 7).Functionally, family II differs from family I in its preference for Mn 2ϩ over Mg 2ϩ as a cofactor. Mg 2ϩ is a better cofactor for family I PPases, and it also activates family II PPases to the same extent. However, Mn 2ϩ co...

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Section: Catalytic Characteristics Of Magnesium-activated Mtppase Andsupporting

confidence: 77%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

An Unusual, His-dependent Family I Pyrophosphatase from Mycobacterium tuberculosis

Tammenkoski

Benini

Magretova

et al. 2005

Journal of Biological Chemistry

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“…23,24 Inorganic pyrophosphatase, purine nucleotide phosphorylase, and glyceraldehyde-3-phosphate dehydrogenase are multimeric enzymes that show cooperativity in their catalysis. [25][26][27] Survival of these proteins suggests that these dynamic processes do not necessarily result in proteolytically-susceptible conformations.…”

Section: Structures Of Survivorsmentioning

confidence: 99%

Energetics-based Protein Profiling on a Proteomic Scale: Identification of Proteins Resistant to Proteolysis

Park

Zhou

Gilmore

et al. 2007

Journal of Molecular Biology

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SUMMARYNative states of proteins are flexible, populating more than just the unique native conformation. The energetics and dynamics resulting from this conformational ensemble are inherently linked to protein function and regulation. Proteolytic susceptibility is one feature determined by this conformational energy landscape. As an attempt to investigate energetics of proteins on a proteomic scale, we challenged the E. coli proteome with extensive proteolysis and determined which proteins, if any, have optimized their energy landscape for resistance to proteolysis. To our surprise, multiple soluble proteins survived the challenge. Maltose binding protein, a survivor from thermolysin digestion, was characterized by in vitro biophysical studies to identify the physical origin of proteolytic resistance. This experimental characterization shows that kinetic stability is responsible for the unusual resistance in maltose binding protein. The biochemical functions of the identified survivors suggest that many of these proteins may have evolved extreme proteolytic resistance because of their critical roles under stressed conditions. Our results suggest that under functional selection proteins can evolve extreme proteolysis resistance by modulating their conformational energy landscapes without the need to invent new folds, and that proteins can be profiled on a proteomic scale according to their energetic properties by using proteolysis as a structural probe.

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“…At ambient temperature, PPase crystals usually diffract to 2.0±1.8 A Ê resolution (Kankare et al, 1996;Harutyunyan et al, 1997;Avaeva et al, 1998). Using¯ash-cooling with mother liquor containing 27±30%(w/v) glycerol providing cryoprotection led to a minor improvement in resolution, to 1.8±1.7 A Ê .…”

Section: Introductionmentioning

confidence: 99%

Improving the X-ray resolution by reversible flash-cooling combined with concentration screening, as exemplified with PPase

Samygina¹,

Antonyuk²,

Lamzin³

et al. 2000

Acta Crystallogr D Biol Cryst

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A signi®cant improvement in the X-ray resolution of crystals of Escherichia coli inorganic pyrophosphatase at cryotemperature was obtained as a result of studying the relationship between the crystal order and cryosolution component concentrations. To perform the experiments, the ability to reverse the¯ash-cooling process and to return a crystal to ambient temperature was used. In each cycle, the crystal was transferred from a cold nitrogen-gas stream to a cryosolution with modi®ed concentrations of the components. The crystal was then¯ash-cooled again and the diffraction quality checked. Such a technique allows the screening of a wide concentration range rather quickly without using a large number of crystals and allows the determination of optimal cryosolution component concentrations. The resolution limit for crystals of pyrophosphatase increased by almost 0.7 A Ê , from 1.8 to 1.15 A Ê .

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Crystal structure of Escherichia coli inorganic pyrophosphatase complexed with SO₄²⁻

Cited by 30 publications

References 12 publications

An Unusual, His-dependent Family I Pyrophosphatase from Mycobacterium tuberculosis

An Unusual, His-dependent Family I Pyrophosphatase from Mycobacterium tuberculosis

Energetics-based Protein Profiling on a Proteomic Scale: Identification of Proteins Resistant to Proteolysis

Improving the X-ray resolution by reversible flash-cooling combined with concentration screening, as exemplified with PPase

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