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Asp-89: a critical residue in maintaining the oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase.
Biochem J. 1999 Oct 01; 343 Pt 1:257-63.BJ

Abstract

Aspartate residues function as proton acceptors in catalysis and are involved in ionic interactions stabilizing subunit assembly. In an attempt to unravel the role of a conserved aspartate (D89) in sheep-liver tetrameric serine hydroxymethyltransferase (SHMT), it was converted into aspargine by site-directed mutagenesis. The purified D89N mutant enzyme had a lower specific activity compared with the wild-type enzyme. It was a mixture of dimers and tetramers with the proportion of tetramers increasing with an increase in the pyridoxal-5'-phosphate (PLP) concentration used during purification. The D89N mutant tetramer was as active as the wild-type enzyme and had similar kinetic and spectral properties in the presence of 500 microM PLP. The quinonoid spectral intermediate commonly seen in the case of SHMT was also seen in the case of D89N mutant tetramer, although the amount of intermediate formed was lower. Although the purified dimer exhibited visible absorbance at 425 nm, it had a negligible visible CD spectrum at 425 nm and was only 5% active. The apo-D89N mutant tetramer was a dimer unlike the apo-form of the wild-type enzyme which was present predominantly as a tetramer. Furthermore the apo mutant dimer could not be reconstituted to the holo-form by the addition of excess PLP, suggesting that dimer-dimer interactions are weak in this mutant. The recently published crystal structure of human liver cytosolic recombinant SHMT indicates that this residue (D90 in the human enzyme) is located at the N-terminal end of the fourth helix of one subunit and packs against K39 from the second N-terminal helix of the other symmetry related subunit forming the tight dimer. D89 is at the interface of tight dimers where the PLP 5'-phosphate is also bound. Mutation of D89 could lead to weakened ionic interactions in the tight dimer interface, resulting in decreased affinity of the enzyme for the cofactor.

Authors+Show Affiliations

Department of Biochemistry, Indian Institute of Science, Bangalore-560012, India.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

10493937

Citation

Krishna Rao, J V., et al. "Asp-89: a Critical Residue in Maintaining the Oligomeric Structure of Sheep Liver Cytosolic Serine Hydroxymethyltransferase." The Biochemical Journal, vol. 343 Pt 1, 1999, pp. 257-63.
Krishna Rao JV, Jagath JR, Sharma B, et al. Asp-89: a critical residue in maintaining the oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase. Biochem J. 1999;343 Pt 1:257-63.
Krishna Rao, J. V., Jagath, J. R., Sharma, B., Appaji Rao, N., & Savithri, H. S. (1999). Asp-89: a critical residue in maintaining the oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase. The Biochemical Journal, 343 Pt 1, 257-63.
Krishna Rao JV, et al. Asp-89: a Critical Residue in Maintaining the Oligomeric Structure of Sheep Liver Cytosolic Serine Hydroxymethyltransferase. Biochem J. 1999 Oct 1;343 Pt 1:257-63. PubMed PMID: 10493937.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Asp-89: a critical residue in maintaining the oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase. AU - Krishna Rao,J V, AU - Jagath,J R, AU - Sharma,B, AU - Appaji Rao,N, AU - Savithri,H S, PY - 1999/9/24/pubmed PY - 1999/9/24/medline PY - 1999/9/24/entrez SP - 257 EP - 63 JF - The Biochemical journal JO - Biochem. J. VL - 343 Pt 1 N2 - Aspartate residues function as proton acceptors in catalysis and are involved in ionic interactions stabilizing subunit assembly. In an attempt to unravel the role of a conserved aspartate (D89) in sheep-liver tetrameric serine hydroxymethyltransferase (SHMT), it was converted into aspargine by site-directed mutagenesis. The purified D89N mutant enzyme had a lower specific activity compared with the wild-type enzyme. It was a mixture of dimers and tetramers with the proportion of tetramers increasing with an increase in the pyridoxal-5'-phosphate (PLP) concentration used during purification. The D89N mutant tetramer was as active as the wild-type enzyme and had similar kinetic and spectral properties in the presence of 500 microM PLP. The quinonoid spectral intermediate commonly seen in the case of SHMT was also seen in the case of D89N mutant tetramer, although the amount of intermediate formed was lower. Although the purified dimer exhibited visible absorbance at 425 nm, it had a negligible visible CD spectrum at 425 nm and was only 5% active. The apo-D89N mutant tetramer was a dimer unlike the apo-form of the wild-type enzyme which was present predominantly as a tetramer. Furthermore the apo mutant dimer could not be reconstituted to the holo-form by the addition of excess PLP, suggesting that dimer-dimer interactions are weak in this mutant. The recently published crystal structure of human liver cytosolic recombinant SHMT indicates that this residue (D90 in the human enzyme) is located at the N-terminal end of the fourth helix of one subunit and packs against K39 from the second N-terminal helix of the other symmetry related subunit forming the tight dimer. D89 is at the interface of tight dimers where the PLP 5'-phosphate is also bound. Mutation of D89 could lead to weakened ionic interactions in the tight dimer interface, resulting in decreased affinity of the enzyme for the cofactor. SN - 0264-6021 UR - https://www.unboundmedicine.com/medline/citation/10493937/Asp_89:_a_critical_residue_in_maintaining_the_oligomeric_structure_of_sheep_liver_cytosolic_serine_hydroxymethyltransferase_ L2 - https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/10493937/ DB - PRIME DP - Unbound Medicine ER -