Subunit interface mutants of rabbit muscle aldolase form active dimers.


Abstract

We report the construction of subunit interface mutants of rabbit muscle aldolase A with altered quaternary structure. A mutation has been described that causes nonspherocytic hemolytic anemia and produces a thermolabile aldolase (Kishi H et al., 1987, Proc Natl Acad Sci USA 84:8623-8627). The disease arises from substitution of Gly for Asp-128, a residue at the subunit interface of human aldolase A. To elucidate the role of this residue in the highly homologous rabbit aldolase A, site-directed mutagenesis is used to replace Asp-128 with Gly, Ala, Asn, Gln, or Val. Rabbit aldolase D128G purified from Escherichia coli is found to be similar to human D128G by kinetic analysis, CD, and thermal inactivation assays. All of the mutant rabbit aldolases are similar to the wild-type rabbit enzyme in secondary structure and kinetic properties. In contrast, whereas the wild-type enzyme is a tetramer, chemical crosslinking and gel filtration indicate that a new dimeric species exists for the mutants. In sedimentation velocity experiments, the mutant enzymes as mixtures of dimer and tetramer at 4 degrees C. Sedimentation at 20 degrees C shows that the mutant enzymes are > 99.5% dimeric and, in the presence of substrate, that the dimeric species is active. Differential scanning calorimetry demonstrates that Tm values of the mutant enzymes are decreased by 12 degrees C compared to wild-type enzyme. The results indicate that Asp-128 is important for interface stability and suggest that 1 role of the quaternary structure of aldolase is to provide thermostability. Study holds ProTherm entries: 10967, 10968, 10969, 10970, 10971, 10972, 10973, 10974, 10975, 10976, 10977, 10978, 10979 Extra Details: additive : NaN3(0.02 %),transition1 aldolase; quaternary structure; site-directed mutagenesis;,subunit interface; thermolability

Submission Details

ID: JVKHnp2E3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:41 p.m.

Version: 1

Publication Details
Beernink PT;Tolan DR,Protein Sci. (1994) Subunit interface mutants of rabbit muscle aldolase form active dimers. PMID:7833800
Additional Information

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 PDB Entries

Structure ID Release Date Resolution Structure Title
5TLE 2017-10-25 1.58 Fructose-1,6-bisphosphate aldolase from rabbit muscle in complex with the inhibitor 2-phosphate-naphthalene 6-bisphosphonate
3BV4 2008-06-24 1.7 Crystal structure of a rabbit muscle fructose-1,6-bisphosphate aldolase A dimer variant
1ZAI 2005-05-10 1.76 Fructose-1,6-bisphosphate Schiff base intermediate in FBP aldolase from rabbit muscle
1ZAH 2005-05-10 1.8 Fructose-1,6-bisphosphate aldolase from rabbit muscle
3DFQ 2009-04-28 1.82 D33S mutant fructose-1,6-bisphosphate aldolase from rabbit muscle
5F4X 2016-12-28 1.84 Fructose-1,6-bisphosphate aldolase K229M mutant from rabbit muscle
3DFN 2009-04-28 1.86 D33N mutant fructose-1,6-bisphosphate aldolase from rabbit muscle
2QUT 2007-08-28 1.88 Dihydroxyacetone phosphate enamine intermediate in fructose-1,6-bisphosphate aldolase from rabbit muscle
1ZAL 2005-05-10 1.89 Fructose-1,6-bisphosphate aldolase from rabbit muscle in complex with partially disordered tagatose-1,6-bisphosphate, a weak competitive inhibitor
1ZAJ 2005-05-10 1.89 Fructose-1,6-bisphosphate aldolase from rabbit muscle in complex with mannitol-1,6-bisphosphate, a competitive inhibitor
1ADO 1997-12-24 1.9 FRUCTOSE 1,6-BISPHOSPHATE ALDOLASE FROM RABBIT MUSCLE
3DFO 2009-04-28 1.94 Dihydroxyacetone phosphate Schiff base and enamine intermediates in D33N mutant fructose-1,6-bisphosphate aldolase from rabbit muscle
3DFT 2009-04-28 1.94 Phosphate ions in D33S mutant fructose-1,6-bisphosphate aldolase from rabbit muscle
5KY6 2017-06-28 1.94 Human muscle fructose-1,6-bisphosphate aldolase
5TLZ 2017-10-25 1.97 Fructose-1,6-bisphosphate aldolase from rabbit muscle in complex with the inhibitor naphthalene 2,6-bisphosphate
2QUU 2007-08-28 1.98 Dihydroxyacetone phosphate Schiff base intermediate in mutant fructose-1,6-bisphosphate aldolase from rabbit muscle
3B8D 2007-11-13 2.0 Fructose 1,6-bisphosphate aldolase from rabbit muscle
1ALD 1992-01-15 2.0 ACTIVITY AND SPECIFICITY OF HUMAN ALDOLASES
3DFS 2009-04-28 2.03 Dihydroxyacetone phosphate Schiff base intermediate in D33S mutant fructose-1,6-bisphosphate aldolase from rabbit muscle
2OT0 2007-02-27 2.05 Fructose-1,6-bisphosphate aldolase from rabbit muscle in complex with a C-terminal peptide of Wiskott-Aldrich syndrome protein
3DFP 2009-04-28 2.05 Phosphate ions in D33N mutant fructose-1,6-bisphosphate aldolase from rabbit muscle
2OT1 2007-02-27 2.05 Fructose-1,6-bisphosphate aldolase from rabbit muscle in complex with naphthol AS-E phosphate, a competitive inhibitor
3TU9 2011-10-12 2.09 Crystal structure of rabbit muscle aldolase bound with 5-O-methyl mannitol 1,6-phosphate
2ALD 1999-04-20 2.1 HUMAN MUSCLE ALDOLASE
5TLH 2017-10-25 2.2 Fructose-1,6-bisphosphate aldolase from rabbit muscle in complex with the inhibitor 2-naphthol 6-bisphosphonate
3LGE 2010-02-02 2.2 Crystal structure of rabbit muscle aldolase-SNX9 LC4 complex
1EX5 2001-01-24 2.2 FRUCTOSE 1,6-BISPHOSPHATE ALDOLASE FROM RABBIT MUSCLE
2QUV 2007-08-28 2.22 Phosphate ions in fructose-1,6-bisphosphate aldolase from rabbit muscle
5TLW 2017-10-25 2.29 Fructose-1,6-bisphosphate aldolase from rabbit muscle in complex with the inhibitor 1-phosphate-benzene 4-bisphosphonate
6ALD 2000-01-05 2.3 RABBIT MUSCLE ALDOLASE A/FRUCTOSE-1,6-BISPHOSPHATE COMPLEX
1EWD 2001-01-24 2.46 FRUCTOSE 1,6-BISPHOSPHATE ALDOLASE FROM RABBIT MUSCLE
5VY5 2017-06-14 2.6 Rabbit muscle aldolase using 200keV
1EWE 2001-01-24 2.6 Fructose 1,6-Bisphosphate Aldolase from Rabbit Muscle
1J4E 2002-02-13 2.65 FRUCTOSE-1,6-BISPHOSPHATE ALDOLASE COVALENTLY BOUND TO THE SUBSTRATE DIHYDROXYACETONE PHOSPHATE
4ALD 1999-03-02 2.8 HUMAN MUSCLE FRUCTOSE 1,6-BISPHOSPHATE ALDOLASE COMPLEXED WITH FRUCTOSE 1,6-BISPHOSPHATE
6MWQ 2018-11-21 3.0 Single particle cryoEM structure of a DARPin-aldolase platform in complex with GFP

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
97.3 Fructose-bisphosphate aldolase A P05065 ALDOA_RAT
97.8 Fructose-bisphosphate aldolase A A5A6I5 ALDOA_PANTR
98.1 Fructose-bisphosphate aldolase A P05064 ALDOA_MOUSE
98.1 Fructose-bisphosphate aldolase A Q5NVR5 ALDOA_PONAB
98.4 Fructose-bisphosphate aldolase A P04075 ALDOA_HUMAN
100.0 Fructose-bisphosphate aldolase A P00883 ALDOA_RABIT