Analysis of the Active-Site Mechanism of Tyrosyl-DNA Phosphodiesterase I: A Member of the Phospholipase D Superfamily

Abstract

Tyrosyl DNA phosphodiesterase I (Tdp1) is a member of the phospholipase D superfamily and hydrolyzes 3′phospho-DNA adducts via two conserved catalytic histidines, one acting as the lead nucleophile and the second as a general acid/base. Substitution of the second histidine specifically to arginine contributes to the neurodegenerative disease SCAN1. We investigated the catalytic role of this histidine in the yeast protein (His432) using a combination of X-ray crystallography, biochemistry, yeast genetics and theoretical chemistry. The structures of wild type Tdp1 and His432Arg both show a phosphorylated form of the nucleophilic histidine that is not observed in the structure of His432Asn. The phosphohistidine is stabilized in the His432Arg structure by the guanidinium group that also restricts access of a nucleophilic water molecule to the Tdp1-DNA intermediate. Biochemical analyses confirm that His432Arg forms an observable and unique Tdp1-DNA adduct during catalysis. Substitution of His432 by Lys does not affect catalytic activity or yeast phenotype, but substitution with Asn, Gln, Leu, Ala, Ser and Thr all result in severely compromised enzymes and Top1-camptothecin dependent lethality. Surprisingly, His432Asn did not show a stable covalent Tdp1-DNA intermediate which suggests another catalytic defect. Theoretical calculations revealed that the defect resides in the nucleophilic histidine and that the pKa of this histidine is crucially dependent upon the second histidine and the incoming phosphate of the substrate. This represents a unique example of substrate-activated catalysis that applies to the entire phospholipase D superfamily.

Tyrosyl-DNA Phosphodiesterase I Catalytic Mutants Reveal an Alternative Nucleophile That Can Catalyze Substrate Cleavage

“…Saccharomyces cerevisiae strain KWY3 [top1⌬, TDP1] (MAT␣, ura3⌬::LoxP his3⌬200 leu2⌬1 trp1⌬63 top1⌬::HIS5) was generated from FY-250 (MAT␣, ura3-52, his3A200, leu2Al, trp1⌬63) by gene replacement of TOP1 with HIS5 and the ura3-52 allele with LoxP-KAN r -LoxP, followed by Cre-mediated recombination to yield ura3⌬::LoxP (28,29). KWY4 [top1⌬,tdp1⌬] (MAT␣, ura3⌬::LoxP his3⌬200 leu2⌬1 trp1⌬63 tdp1⌬ top1::HIS5) was generated from KWY3 via deletion of TDP1 using URA3 flanked by gene endogenous 3Ј-UTR repeats, followed by selection on 5-fluoroorotic acid (23,30 For cell viability studies, the tdp1 alleles were expressed from the galactose-inducible promoter GAL1 in YCpGAL1TDP1⅐L vectors as described in Gajewski et al (23). tdp1 mutant alleles were created using QuikChange Site-Directed Mutagenesis kit (Stratagene).…”

“…For example, a similar change in electrostatic potential and DNA binding was induced in the H432K mutant; however, this substitution did not impair the resolution of the covalent Tdp1-DNA linkage. Indeed, the catalytic activity of this mutant is similar to that of wild type Tdp1 (23). In contrast, substitution of His gab with residues that contain smaller polar or aliphatic side chains (including Asn, Glu, Leu, Ala, Ser, or Thr) induces an even more severe lethal phenotype, effectively transforming Tdp1 into a potent Top1-dependent cellular toxin, even in the absence of CPT (7,23).…”

“…In structural studies of the analogous SCAN1-like mutation in yeast Tdp1 (H432R), crystallographic data revealed a more shallow catalytic pocket with increased positive charge, in comparison with wild type Tdp1 (23). Such alterations in electrostatic potential appear to enhance the noncovalent binding of Tdp1 to DNA.…”

“…Replacing His gab with Arg impairs the resolution of the Tdp1-DNA phosphohistidyl linkage, resulting in a longer-lived covalent Tdp1-DNA reaction intermediate in vitro (10,23). Consequently, SCAN1 lymphoblastoid cells or yeast cells expressing the analogous yeast His gab to Arg (H432R) mutant are more sensitive to CPT-induced DNA lesions or to oxidative DNA damage induced by H 2 O 2 , ␥-irradiation, or bleomycin (7,(23)(24)(25). However, in patients, the SCAN1 phenotype is not evident until the second decade of life, is restricted primarily to the neurodegeneration of cerebellar neurons, and is not associated with an increased risk of cancer or immune deficiency (13).…”

“…For example, mutation of the His nuc to Ala in human and yeast Tdp1 has been reported to inhibit Tdp1 catalytic activity in in vitro assays (12,16,26,27). We therefore hypothesized that introducing the His nuc Ala mutation into the His gab mutant proteins would suppress the enhanced CPT sensitivity of the SCAN1 H432R mutant and the Top1-dependent lethality of the more toxic H432N mutant (7,23,24). Surprisingly, however, we report here that the His nuc Ala substitution (H182A) actually potentiated the cytotoxicity of the His gab mutants and induced a Top1-independent lethal phenotype in combination with the H432N mutant.…”