Functional Characterisation of Three Glycine N-Acyltransferase Variants and the Effect on Glycine Conjugation to Benzoyl–CoA
Abstract
:1. Introduction
2. Results and Discussion
2.1. Level of Conservation of the GLYAT Gene
2.2. Relative Enzyme Activity and Catalytic Parameters
2.3. Comparison of GLYAT Kinetic Parameters to Literature Values
3. Materials and Methods
3.1. Missense Variants Identified in GLYAT Using gnomAD
3.2. Haplotype Data Obtained from Ensembl
3.3. Tajima’s Test of Neutrality
3.4. Phylogenetic Analyses
3.5. Expression and Nickel-Affinity Purification of the Recombinant GLYAT Haplotypes
3.6. Bi-Substrate (Benzoyl–CoA and Glycine) Kinetic Analysis
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Population | Exomes | Genomes | Total |
---|---|---|---|
African/African American | 8 128 | 4 359 | 12,487 |
Latino | 17,296 | 424 | 17,720 |
Ashkenazi Jewish | 5 040 | 145 | 5 185 |
East Asian | 9 197 | 780 | 9 977 |
Finnish | 10,824 | 1 738 | 12,562 |
Non-Finnish European | 56,885 | 7 718 | 64,603 |
South Asian | 15,308 | # | 15,308 |
Other* | 3 070 | 544 | 3 614 |
Female | 57,787 | 6 967 | 64,754 |
Male | 67,961 | 8 741 | 76,702 |
Total | 125,748 | 15,708 | 138,632 |
Gene | Number of Sequences (m) | Number of Segregating Sites (S) | Nucleotide Diversity (π) | Tajima Test Statistic (D) |
---|---|---|---|---|
GLYAT | 25 | 21 | 0.007748 | −2.13 |
Haplotype | Vf µmol min−1 mg protein−1 | kcat s−1 | s0.5,gly mM | hgly | s0.5,benz µM | hbenz |
---|---|---|---|---|---|---|
156Asn > Ser | 0.85 ± 0.06 | 0.48 ± 0.03 | 23 ± 2 | 1.6 ± 0.1 | 97 ± 3 | 2.1 ± 0.1 |
17Ser > Thr,156Asn > Ser | 0.62 ± 0.02 | 0.35 ± 0.01 | 29 ± 3 | 1.3 ± 0.1 | 118 ± 7 | 1.5 ± 0.1 |
156Asn > Ser,199Arg > Cys | 0.083 ± 0.005 | 0.047 ± 0.003 | 30 ± 3 | 1.4 ± 0.1 | 61 ± 3 | 3.5 ± 0.5 |
Parameters | Values | Recombinant Variant/Isolated from Liver | Reference |
---|---|---|---|
KMapp (benzoyl-CoA) (µM) | 13 | Purified GLYAT from human liver | [33] |
28 ± 5 | Purified recombinant 17Ser > Thr variant | [31] | |
38 ± 4 | Purified recombinant 156Asn > Ser variant | [31] | |
s0.5 | 61 ± 3 | Purified recombinant 156Asn > Ser,199Arg > Cys variant | This study (bi-substrate Hill) |
KMapp (benzoyl-CoA) (µM) | 67 ± 5 | Partially purified GLYAT from human liver | [32] |
79 ± 38 | Purified recombinant wildtype | [34] | |
88 ± 66 | Purified recombinant 156Asn > Ser variant | [34] | |
s0.5 | 97 ± 3 | Purified recombinant 156Asn > Ser variant | This study (bi-substrate Hill) |
118 ± 7 | Purified recombinant 17Ser > Thr,156Asn > Ser variant | This study (bi-substrate Hill) | |
KMapp (benzoyl-CoA) (µM) | 139 ± 85 | Purified recombinant L61 variant | [34] |
209 | Purified recombinant 156Asn > Ser variant | [28] | |
57900 * | Purified GLYAT from human liver | [30] | |
KMapp (glycine) (mM) | 6.4 | Purified GLYAT from human liver | [33] |
6.5 ± 1 | Partially purified GLYAT from human liver | [32] | |
s0.5 | 23 ± 2 | Purified recombinant 156Asn > Ser variant | This study (bi-substrate Hill) |
KMapp (glycine) (mM) | 26.6 | Purified recombinant 156Asn > Ser variant | [28] |
s0.5 | 29 ± 3 | Purified recombinant 17Ser > Thr,156Asn > Ser variant | This study (bi-substrate Hill) |
30 ± 3 | Purified recombinant 156Asn > Ser199Arg > Cys variant | This study (bi-substrate Hill) | |
Vmax (nmol/min/mg) | 83 ± 5 | Purified recombinant 156Asn > Ser199Arg > Cys variant | This study (bi-substrate Hill) |
543 ± 21 | Purified GLYAT from human liver | [33] | |
620 ± 20 | Purified recombinant 17Ser > Thr,156Asn > Ser variant | This study (bi-substrate Hill) | |
665 ± 40 | Purified recombinant 17Ser > Thr variant | [31] | |
807 | Recombinant 156Asn > Ser variant | [28] | |
850 ± 60 | Purified recombinant 156Asn > Ser variant | This study (bi-substrate Hill) | |
1230 | Purified recombinant 156Asn > Ser variant | [31] | |
17,100 # | Purified GLYAT from human liver | [30] | |
121,000 + | Purified recombinant L61 variant | [34] | |
490,000 + | Purified recombinant wildtype | [34] | |
1,359,000 + | Purified recombinant 156Asn > Ser variant | [34] |
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Rohwer, J.M.; Schutte, C.; van der Sluis, R. Functional Characterisation of Three Glycine N-Acyltransferase Variants and the Effect on Glycine Conjugation to Benzoyl–CoA. Int. J. Mol. Sci. 2021, 22, 3129. https://doi.org/10.3390/ijms22063129
Rohwer JM, Schutte C, van der Sluis R. Functional Characterisation of Three Glycine N-Acyltransferase Variants and the Effect on Glycine Conjugation to Benzoyl–CoA. International Journal of Molecular Sciences. 2021; 22(6):3129. https://doi.org/10.3390/ijms22063129
Chicago/Turabian StyleRohwer, Johann M., Chantelle Schutte, and Rencia van der Sluis. 2021. "Functional Characterisation of Three Glycine N-Acyltransferase Variants and the Effect on Glycine Conjugation to Benzoyl–CoA" International Journal of Molecular Sciences 22, no. 6: 3129. https://doi.org/10.3390/ijms22063129