Transition-State Analysis of the DNA Repair Enzyme MutY

174

Adenine DNA glycosylase catalyzes the glycolytic removal of adenine from the promutagenic A⅐oxoG base pair in DNA. The general features of DNA recognition by an adenine DNA glycosylase, Bacillus stearothermophilus MutY, have previously been revealed via the X-ray structure of a catalytically inactive mutant protein bound to an A:oxoG-containing DNA duplex. Although the structure revealed the substrate adenine to be, as expected, extruded from the DNA helix and inserted into an extrahelical active site pocket on the enzyme, the substrate adenine engaged in no direct contacts with active site residues. This feature was paradoxical, because other glycosylases have been observed to engage their substrates primarily through direct contacts. The lack of direct contacts in the case of MutY suggested that either MutY uses a distinctive logic for substrate recognition or that the X-ray structure had captured a noncatalytically competent state in lesion recognition. To gain further insight into this issue, we crystallized wild-type MutY bound to DNA containing a catalytically inactive analog of 2-deoxyadenosine in which a single 2-H atom was replaced by fluorine. The structure of this fluorinated lesionrecognition complex (FLRC) reveals the substrate adenine buried more deeply into the active site pocket than in the prior structure and now engaged in multiple direct hydrogen bonding and hydrophobic interactions. This structure appears to capture the catalytically competent state of adenine DNA glycosylases, and it suggests a catalytic mechanism for this class of enzymes, one in which general acid-catalyzed protonation of the nucleobase promotes glycosidic bond cleavage.base-excision repair ͉ general acid catalysis ͉ substrate recognition T he genotoxic DNA lesion 8-oxoguanine (oxoG) arises chronically in cells through the attack of endogenous electrophilic oxidants on guanine residues (Fig. 1A). Left unrepaired, these oxoG lesions mispair with A during DNA replication, thereby giving rise to G⅐C to T⅐A transversion mutations. Most organisms possess a repair system dedicated to countering the deleterious effects of oxoG. The so-called GO system in eubacteria (1, 2) (Fig. 1B), for example, consists of proteins that directly sanitize the nucleotide precursor pool (MutT) and DNA (MutM or Fpg) of oxoG residues. Proteins that serve functions equivalent to those of MutT and MutM are found in all eukaryotic organisms (3). Failure of this first round of defense results in replicative production of oxoG:A pairs (4), which are particularly troublesome to repair, with neither nucleobase providing faithful information with which to direct correction of the other: oxoG does not belong in DNA at all, and although A is a canonical nucleobase, it is contextually aberrant in the oxoG:A pair because it replaces what should be a C. The multistep repair of oxoG:A is initiated by the highly evolutionarily conserved enzyme adenine DNA glycosylase (MutY in bacteria, hMYH in humans), which catalyzes hydrolysis of the glycosidic linkage between th...

Section: General Features Of the Muty Flrcsupporting

confidence: 74%

Section: General Features Of the Muty Flrcmentioning

confidence: 99%

Atomic substitution reveals the structural basis for substrate adenine recognition and removal by adenine DNA glycosylase

Lee

Verdine

2009

174

“…2C) and indicate that the gap is reduced by electrophile migration after cleavage of the base-sugar bond (36). Notably, our structure indicates that the backbone carbonyl oxygen of Asn140 is well positioned to stabilize the glycosyl cation that is generated upon base-sugar bond cleavage (37,38).…”

Section: Resultsmentioning

confidence: 83%

Lesion processing by a repair enzyme is severely curtailed by residues needed to prevent aberrant activity on undamaged DNA

Maiti

Noon

MacKerell

et al. 2012

168

DNA base excision repair is essential for maintaining genomic integrity and for active DNA demethylation, a central element of epigenetic regulation. A key player is thymine DNA glycosylase (TDG), which excises thymine from mutagenic G·T mispairs that arise by deamination of 5-methylcytosine (mC). TDG also removes 5-formylcytosine and 5-carboxylcytosine, oxidized forms of mC produced by Tet enzymes. Recent studies show that the glycosylase activity of TDG is essential for active DNA demethylation and for embryonic development. Our understanding of how repair enzymes excise modified bases without acting on undamaged DNA remains incomplete, particularly for mismatch glycosylases such as TDG. We solved a crystal structure of TDG (catalytic domain) bound to a substrate analog and characterized active-site residues by mutagenesis, kinetics, and molecular dynamics simulations. The studies reveal how TDG binds and positions the nucleophile (water) and uncover a previously unrecognized catalytic residue (Thr197). Remarkably, mutation of two active-site residues (Ala145 and His151) causes a dramatic enhancement in G·T glycosylase activity but confers even greater increases in the aberrant removal of thymine from normal A·T base pairs. The strict conservation of these residues may reflect a mechanism used to strike a tolerable balance between the requirement for efficient repair of G·T lesions and the need to minimize aberrant action on undamaged DNA, which can be mutagenic and cytotoxic. Such a compromise in G·T activity can account in part for the relatively weak G·T activity of TDG, a trait that could potentially contribute to the hypermutability of CpG sites in cancer and genetic disease. D NA base excision repair (BER) plays an established role in maintaining genomic integrity, and recent studies indicate that BER is also essential for active DNA demethylation, a key element of epigenetic gene regulation (1-3). A central player in both processes is thymine DNA glycosylase (TDG), which initiates BER by excising damaged or modified forms of 5-methylcytosine (mC) that arise at 5′-CpG-3′ sites. TDG was discovered for its ability to selectively remove T from G·T mispairs, mutagenic lesions that can arise from deamination of mC to T (4, 5). TDG also excises 5-formylcytosine (fC) and 5-carboxylcytosine (caC), oxidized forms of mC that are generated by Tet enzymes (6, 7). In addition, TDG was shown to be essential for active DNA demethylation and for embryonic development (8, 9), a role that likely involves excision of deaminated or oxidized forms of mC generated by a deaminase or Tet enzyme (7,(10)(11)(12). Thus, the ability of TDG to remove deaminated and oxidized forms of mC is important for genetic and epigenetic integrity.The question of how DNA glycosylases remove modified bases without acting upon the huge excess of undamaged DNA remains largely unresolved, particularly for mismatch glycosylases such as TDG and MBD4 (methyl binding domain IV) (13-15). These enzymes face the formidable task of removing a normal base, t...

“…Intrinsic KIEs for labeled inosines as substrates for PNP reflect differences in bond vibrational environment between substrates in solution and at the transition state (24). Accordingly, KIE analysis has been used to probe transition-state structures of PNP as well as several related enzyme-catalyzed reactions (6,(27)(28)(29)(30). The intrinsic KIEs of heavy and light PNP are indistinguishable within experimental error ( Table 1), indicating that altered femtosecond protein dynamic motions do not change the geometry of the PNP transition state.…”

Section: Discussionmentioning

confidence: 99%

Femtosecond dynamics coupled to chemical barrier crossing in a Born-Oppenheimer enzyme

Silva

Murkin

Schramm

2011

114

204

Contributions of fast (femtosecond) dynamic motion to barrier crossing at enzyme catalytic sites is in dispute. Human purine nucleoside phosphorylase (PNP) forms a ribocation-like transition state in the phosphorolysis of purine nucleosides and fast protein motions have been proposed to participate in barrier crossing. In the present study, 13 C−, 15 N−, 2 H−labeled human PNP (heavy PNP) was expressed, purified to homogeneity, and shown to exhibit a 9.9% increase in molecular mass relative to its unlabeled counterpart (light PNP). Kinetic isotope effects and steady-state kinetic parameters were indistinguishable for both enzymes, indicating that transition-state structure, equilibrium binding steps, and the rate of product release were not affected by increased protein mass. Single-turnover rate constants were slowed for heavy PNP, demonstrating reduced probability of chemical barrier crossing from enzyme-bound substrates to enzyme-bound products. In a second, independent method to probe barrier crossing, heavy PNP exhibited decreased forward commitment factors, also revealing mass-dependent decreased probability for barrier crossing. Increased atomic mass in human PNP alters bond vibrational modes on the femtosecond time scale and reduces on-enzyme chemical barrier crossing. This study demonstrates coupling of enzymatic bond vibrations on the femtosecond time scale to barrier crossing. femtosecond motions | kinetic isotope effect | transition state structure | enzymatic catalysis | protein dynamics