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Importance of the amino terminus in maintenance of oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase.
Eur J Biochem. 1997 Jul 01; 247(1):372-9.EJ

Abstract

The role of the amino and carboxyl-terminal regions of cytosolic serine hydroxymethyltransferase (SHMT) in subunit assembly and catalysis was studied using six amino-terminal (lacking the first 6, 14, 30, 49, 58, and 75 residues) and two carboxyl-terminal (lacking the last 49 and 185 residues) deletion mutants. These mutants were constructed from a full length cDNA clone using restriction enzyme/PCR-based methods and overexpressed in Escherichia coli. The overexpressed proteins, des-(A1-K6)-SHMT and des-(A1-W14)-SHMT were present in the soluble fraction and they were purified to homogeneity. The deletion clones, for des-(A1-V30)-SHMT and des-(A1-L49)-SHMT were expressed at very low levels, whereas des-(A1-R58)-SHMT, des-(A1-G75)-SHMT, des-(Q435-F483)-SHMT and des-(L299-F483)-SHMT mutant proteins were not soluble and formed inclusion bodies. Des-(A1-K6)-SHMT and des-(A1-W14)-SHMT catalyzed both the tetrahydrofolate-dependent and tetrahydrofolate-independent reactions, generating characteristic spectral intermediates with glycine and tetrahydrofolate. The two mutants had similar kinetic parameters to that of the recombinant SHMT (rSHMT). However, at 55 degrees C, the des-(A1-W14)-SHMT lost almost all the activity within 5 min, while at the same temperature rSHMT and des-(A1-K6)-SHMT retained 85% and 70% activity, respectively. Thermal denaturation studies showed that des-(A1-W14)-SHMT had a lower apparent melting temperature (52 degrees C) compared to rSHMT (56 degrees C) and des-(A1-K6)-SHMT (55 degrees C), suggesting that N-terminal deletion had resulted in a decrease in the thermal stability of the enzyme. Further, urea induced inactivation of the enzymes revealed that 50% inactivation occurred at a lower urea concentration (1.2+/-0.1 M) in the case of des-(A1-W14)-SHMT compared to rSHMT (1.8+/-0.1 M) and des-(A1-K6)-SHMT (1.7+/-0.1 M). The apoenzyme of des-(A1-W14)-SHMT was present predominantly in the dimer form, whereas the apoenzymes of rSHMT and des-(A1-K6)-SHMT were a mixture of tetramers (approximately 75% and approximately 65%, respectively) and dimers. While, rSHMT and des-(A1-K6)-SHMT apoenzymes could be reconstituted upon the addition of pyridoxal-5'-phosphate to 96% and 94% enzyme activity, respectively, des-(A1-W14)-SHMT apoenzyme could be reconstituted only up to 22%. The percentage activity regained correlated with the appearance of visible CD at 425 nm and with the amount of enzyme present in the tetrameric form upon reconstitution as monitored by gel filtration. These results demonstrate that, in addition to the cofactor, the N-terminal arm plays an important role in stabilizing the tetrameric structure of SHMT.

Authors+Show Affiliations

Department of Biochemistry, Indian Institute of Science, Bangalore.No affiliation info availableNo 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

9249049

Citation

Jagath, J R., et al. "Importance of the Amino Terminus in Maintenance of Oligomeric Structure of Sheep Liver Cytosolic Serine Hydroxymethyltransferase." European Journal of Biochemistry, vol. 247, no. 1, 1997, pp. 372-9.
Jagath JR, Sharma B, Bhaskar B, et al. Importance of the amino terminus in maintenance of oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase. Eur J Biochem. 1997;247(1):372-9.
Jagath, J. R., Sharma, B., Bhaskar, B., Datta, A., Rao, N. A., & Savithri, H. S. (1997). Importance of the amino terminus in maintenance of oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase. European Journal of Biochemistry, 247(1), 372-9.
Jagath JR, et al. Importance of the Amino Terminus in Maintenance of Oligomeric Structure of Sheep Liver Cytosolic Serine Hydroxymethyltransferase. Eur J Biochem. 1997 Jul 1;247(1):372-9. PubMed PMID: 9249049.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Importance of the amino terminus in maintenance of oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase. AU - Jagath,J R, AU - Sharma,B, AU - Bhaskar,B, AU - Datta,A, AU - Rao,N A, AU - Savithri,H S, PY - 1997/7/1/pubmed PY - 1997/7/1/medline PY - 1997/7/1/entrez SP - 372 EP - 9 JF - European journal of biochemistry JO - Eur. J. Biochem. VL - 247 IS - 1 N2 - The role of the amino and carboxyl-terminal regions of cytosolic serine hydroxymethyltransferase (SHMT) in subunit assembly and catalysis was studied using six amino-terminal (lacking the first 6, 14, 30, 49, 58, and 75 residues) and two carboxyl-terminal (lacking the last 49 and 185 residues) deletion mutants. These mutants were constructed from a full length cDNA clone using restriction enzyme/PCR-based methods and overexpressed in Escherichia coli. The overexpressed proteins, des-(A1-K6)-SHMT and des-(A1-W14)-SHMT were present in the soluble fraction and they were purified to homogeneity. The deletion clones, for des-(A1-V30)-SHMT and des-(A1-L49)-SHMT were expressed at very low levels, whereas des-(A1-R58)-SHMT, des-(A1-G75)-SHMT, des-(Q435-F483)-SHMT and des-(L299-F483)-SHMT mutant proteins were not soluble and formed inclusion bodies. Des-(A1-K6)-SHMT and des-(A1-W14)-SHMT catalyzed both the tetrahydrofolate-dependent and tetrahydrofolate-independent reactions, generating characteristic spectral intermediates with glycine and tetrahydrofolate. The two mutants had similar kinetic parameters to that of the recombinant SHMT (rSHMT). However, at 55 degrees C, the des-(A1-W14)-SHMT lost almost all the activity within 5 min, while at the same temperature rSHMT and des-(A1-K6)-SHMT retained 85% and 70% activity, respectively. Thermal denaturation studies showed that des-(A1-W14)-SHMT had a lower apparent melting temperature (52 degrees C) compared to rSHMT (56 degrees C) and des-(A1-K6)-SHMT (55 degrees C), suggesting that N-terminal deletion had resulted in a decrease in the thermal stability of the enzyme. Further, urea induced inactivation of the enzymes revealed that 50% inactivation occurred at a lower urea concentration (1.2+/-0.1 M) in the case of des-(A1-W14)-SHMT compared to rSHMT (1.8+/-0.1 M) and des-(A1-K6)-SHMT (1.7+/-0.1 M). The apoenzyme of des-(A1-W14)-SHMT was present predominantly in the dimer form, whereas the apoenzymes of rSHMT and des-(A1-K6)-SHMT were a mixture of tetramers (approximately 75% and approximately 65%, respectively) and dimers. While, rSHMT and des-(A1-K6)-SHMT apoenzymes could be reconstituted upon the addition of pyridoxal-5'-phosphate to 96% and 94% enzyme activity, respectively, des-(A1-W14)-SHMT apoenzyme could be reconstituted only up to 22%. The percentage activity regained correlated with the appearance of visible CD at 425 nm and with the amount of enzyme present in the tetrameric form upon reconstitution as monitored by gel filtration. These results demonstrate that, in addition to the cofactor, the N-terminal arm plays an important role in stabilizing the tetrameric structure of SHMT. SN - 0014-2956 UR - https://www.unboundmedicine.com/medline/citation/9249049/Importance_of_the_amino_terminus_in_maintenance_of_oligomeric_structure_of_sheep_liver_cytosolic_serine_hydroxymethyltransferase_ L2 - https://onlinelibrary.wiley.com/resolve/openurl?genre=article&sid=nlm:pubmed&issn=0014-2956&date=1997&volume=247&issue=1&spage=372 DB - PRIME DP - Unbound Medicine ER -