TY - JOUR

T1 - The Role of Key Amino Acids of the Human Fe(II)/2OG-Dependent Dioxygenase ALKBH3 in Structural Dynamics and Repair Activity toward Methylated DNA

AU - Kanazhevskaya, Lyubov Yu

AU - Gorbunov, Alexey A

AU - Lukina, Maria V

AU - Smyshliaev, Denis A

AU - Zhdanova, Polina V

AU - Lomzov, Alexander A

AU - Koval, Vladimir V

N1 - This work was supported by Russian Science Foundation grant No. 22-24-00699.

PY - 2024/1

Y1 - 2024/1

N2 - Non-heme dioxygenases of the AlkB family hold a unique position among enzymes that repair alkyl lesions in nucleic acids. These enzymes activate the Fe(II) ion and molecular oxygen through the coupled decarboxylation of the 2-oxoglutarate co-substrate to subsequently oxidize the substrate. ALKBH3 is a human homolog of E. coli AlkB, which displays a specific activity toward N1-methyladenine and N3-methylcytosine bases in single-stranded DNA. Due to the lack of a DNA-bound structure of ALKBH3, the basis of its substrate specificity and structure-function relationships requires further exploration. Here we have combined biochemical and biophysical approaches with site-directed mutational analysis to elucidate the role of key amino acids in maintaining the secondary structure and catalytic activity of ALKBH3. Using stopped-flow fluorescence spectroscopy we have shown that conformational dynamics play a crucial role in the catalytic repair process catalyzed by ALKBH3. A transient kinetic mechanism, which comprises the steps of the specific substrate binding, eversion, and anchoring within the DNA-binding cleft, has been described quantitatively by rate and equilibrium constants. Through CD spectroscopy, we demonstrated that replacing side chains of Tyr143, Leu177, and His191 with alanine results in significant alterations in the secondary structure content of ALKBH3 and decreases the stability of mutant proteins. The bulky side chain of Tyr143 is critical for binding the methylated base and stabilizing its flipped-out conformation, while its hydroxyl group is likely involved in facilitating the product release. The removal of the Leu177 and His191 side chains substantially affects the secondary structure content and conformational flexibility, leading to the complete inactivation of the protein. The mutants lacking enzymatic activity exhibit a marked decrease in antiparallel β-strands, offset by an increase in the helical component.

AB - Non-heme dioxygenases of the AlkB family hold a unique position among enzymes that repair alkyl lesions in nucleic acids. These enzymes activate the Fe(II) ion and molecular oxygen through the coupled decarboxylation of the 2-oxoglutarate co-substrate to subsequently oxidize the substrate. ALKBH3 is a human homolog of E. coli AlkB, which displays a specific activity toward N1-methyladenine and N3-methylcytosine bases in single-stranded DNA. Due to the lack of a DNA-bound structure of ALKBH3, the basis of its substrate specificity and structure-function relationships requires further exploration. Here we have combined biochemical and biophysical approaches with site-directed mutational analysis to elucidate the role of key amino acids in maintaining the secondary structure and catalytic activity of ALKBH3. Using stopped-flow fluorescence spectroscopy we have shown that conformational dynamics play a crucial role in the catalytic repair process catalyzed by ALKBH3. A transient kinetic mechanism, which comprises the steps of the specific substrate binding, eversion, and anchoring within the DNA-binding cleft, has been described quantitatively by rate and equilibrium constants. Through CD spectroscopy, we demonstrated that replacing side chains of Tyr143, Leu177, and His191 with alanine results in significant alterations in the secondary structure content of ALKBH3 and decreases the stability of mutant proteins. The bulky side chain of Tyr143 is critical for binding the methylated base and stabilizing its flipped-out conformation, while its hydroxyl group is likely involved in facilitating the product release. The removal of the Leu177 and His191 side chains substantially affects the secondary structure content and conformational flexibility, leading to the complete inactivation of the protein. The mutants lacking enzymatic activity exhibit a marked decrease in antiparallel β-strands, offset by an increase in the helical component.

KW - Humans

KW - AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/genetics

KW - Amino Acids

KW - DNA

KW - Ferrous Compounds

KW - DNA Methylation

UR - https://www.scopus.com/record/display.uri?eid=2-s2.0-85183258577&origin=inward&txGid=234cafc4949475a864ec2722be6e138e

U2 - 10.3390/ijms25021145

DO - 10.3390/ijms25021145

M3 - Article

C2 - 38256217

VL - 25

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

SN - 1661-6596

IS - 2

M1 - 1145

ER -