The extreme thermostable pyrophosphatase from Sulfolobus acidocaldarius: enzymatic and comparative biophysical characterization.


Abstract

Recombinant pyrophosphatase from the hyperthermophilic archaebacterium Sulfolobus acidocaldarius (S-PPase) has been heterologously expressed in Escherichia coli and could be purified in large quantities. S-PPase, previously described as a tetrameric enzyme, was shown to be a homohexameric protein that had catalytic activity with Mg2+ > Zn2+ > Co2+ > Mn2+ > Ni2+, Ca2+. CD and FTIR spectra demonstrate a similar overall fold for S-PPase and PPases from E. coli (E-PPase) and Thermus thermophilus (T-PPase). The relative proportions of secondary structure elements in S-PPase are close to those of a previously proposed model. S-PPase is extremely heat resistant. Even at 95 degrees C the half-life of catalytic activity is 2.5 h, which is dramatically increased in the presence of divalent cations. More than one Mg2+ per monomer is needed for catalysis, but no more than one Mg2+ per monomer is sufficient for thermal stabilization. The Tm values for S-PPase are 89 degrees C (+EDTA), 99 degrees C (+Mg2+), and >100 degrees C (+Mn2+), compared to 58 degrees C (+EDTA), 84 degrees C (+Mg2+), and 93 degrees C (+Mn2+) for E-PPase and 86 degrees C (+EDTA), 99 degrees C (+Mg2+), and 96 degrees C (+Mn2+) for T-PPase. The guanidium hydrochloride-induced unfolding follows an unknown mechanism with a biphasic kinetic and an unstable intermediate. Unfolding curves of the S-, E-, and T-PPase are independent of the method applied (CD spectroscopy and fluorescence) and show a sigmoidal and monophasic transition, indicating a change in global structure during unfolding, which can be described by a two-state process comprising dissociation and denaturation of the folded hexamer into six monomers. The respective DeltaGN-->D(25 degrees C) values of the three PPases vary from 220 to 290 kJ/mol for the overall process and are not significantly higher for the two thermophilic PPases. The stabilizing effect of Mg2+ DeltaDeltaG(25 degrees C) is 16 kJ/mol for E-PPase and 5.5-8 kJ/mol for S-PPase and T-PPase. Study holds ProTherm entries: 12946, 12947, 12948, 12949, 12950, 12951, 12952, 12953, 12954, 12955, 12956, 12957, 12958, 12959, 12960, 12961, 12962, 12963, 12964, 12965 Extra Details: EDTA(5 mM) was added in the experiment pyrophosphatase; stability GdnHCl; denaturation; Mg2+; hexamer

Submission Details

ID: whvmgAkk3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:44 p.m.

Version: 1

Publication Details
Hansen T;Urbanke C;Leppänen VM;Goldman A;Brandenburg K;Schäfer G,Arch. Biochem. Biophys. (1999) The extreme thermostable pyrophosphatase from Sulfolobus acidocaldarius: enzymatic and comparative biophysical characterization. PMID:10049508
Additional Information

Study Summary

Number of data points 44
Proteins Inorganic pyrophosphatase ; Inorganic pyrophosphatase ; Inorganic pyrophosphatase ; Inorganic pyrophosphatase ; Inorganic pyrophosphatase ; Inorganic pyrophosphatase
Unique complexes 3
Assays/Quantities/Protocols Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O ; Experimental Assay: Cm temp:50.0 C, details:Additives EDTA (5 mM), ; Experimental Assay: m temp:50.0 C, details:Additives EDTA (5 mM), ; Experimental Assay: dG_H2O temp:50.0 C, details:Additives EDTA (5 mM), ; Experimental Assay: Cm temp:50.0 C ; Experimental Assay: m temp:50.0 C ; Experimental Assay: dG_H2O temp:50.0 C ; Experimental Assay: Cm details:Additives EDTA (5 mM), ; Experimental Assay: m details:Additives EDTA (5 mM), ; Experimental Assay: dG_H2O details:Additives EDTA (5 mM), ; Experimental Assay: Cm details:Additives EDTA (5 mM), ; Experimental Assay: m details:Additives EDTA (5 mM), ; Experimental Assay: dG_H2O details:Additives EDTA (5 mM), ; Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O ; Experimental Assay: Tm details:Additives Mn2+(5 mM), ; Experimental Assay: Tm details:Additives Mg2+(5 mM), ; Experimental Assay: Tm ionic:EDTA: 5 mM, details:Additives EDTA (5 mM), ; Experimental Assay: Tm details:Additives EDTA (5 mM),
Libraries Mutations for sequence MKLSPGKNAPDVVNVLVEIPQGSNIKYEYDDEEGVIKVDRVLYTSMNYPFNYGFIPGTLEEDGDPLDVLVITNYQLYPGSVIEVRPIGILYMKDEEGEDAKIVAVPKDKTDPSFSNIKDINDLPQATKNKIVHFFEHYKELEPGKYVKISGWGSATEAKNRIQLAIKRVSGGQ ; Mutations for sequence ANLKSLPVGDKAPEVVHMVIEVPRGSGNKYEYDPDLGAIKLDRVLPGAQFYPGDYGFIPSTLAEDGDPLDGLVLSTYPLLPGVVVEVRVVGLLLMEDEKGGDAKVIGVVAEDQRLDHIQDIGDVPEGVKQEIQHFFETYKALEAKKGKWVKVTGWRDRKAALEEVRACIARYKG ; Mutations for sequence SLLNVPAGKDLPEDIYVVIEIPANADPIKYEIDKESGALFVDRFMSTAMFYPCNYGYINHTLSLDGDPVDVLVPTPYPLQPGSVIRCRPVGVLKMTDEAGEDAKLVAVPHSKLSKEYDHIKDVNDLPELLKAQIAHFFEHYKDLEKGKWVKVEGWENAEAAKAEIVASFERAKNK

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Inorganic pyrophosphatase P0A7B0 IPYR_ECO57
100.0 Inorganic pyrophosphatase P0A7A9 IPYR_ECOLI
98.9 Inorganic pyrophosphatase Q8FAG0 IPYR_ECOL6
94.3 Inorganic pyrophosphatase P65749 IPYR_SALTI
94.3 Inorganic pyrophosphatase P65748 IPYR_SALTY
91.4 Inorganic pyrophosphatase Q8ZB98 IPYR_YERPE
100.0 Inorganic pyrophosphatase P50308 IPYR_SULAC
100.0 Inorganic pyrophosphatase P38576 IPYR_THET8