Messick Troy E, Chmiel Nikolas H, Golinelli Marie-Pierre, Langer Michael R, Joshua-Tor Leemor, David Sheila S
Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.
Biochemistry. 2002 Mar 26;41(12):3931-42. doi: 10.1021/bi012035x.
The Escherichia coli DNA repair enzyme MutY plays an important role in the recognition and repair of 7,8-dihydro-8-oxo-2'-deoxyguanosine-2'-deoxyadenosine (OG*A) mismatches in DNA. MutY prevents DNA mutations caused by the misincorporation of A opposite OG by catalyzing the deglycosylation of the aberrant adenine. MutY is representative of a unique subfamily of DNA repair enzymes that also contain a [4Fe-4S]2+ cluster, which has been implicated in substrate recognition. Previously, we have used site-directed mutagenesis to individually replace the cysteine ligands to the [4Fe-4S]2+ cluster of E. coli MutY with serine, histidine, or alanine. These experiments suggested that histidine coordination to the iron-sulfur cluster may be accommodated in MutY at position 199. Purification and enzymatic analysis of C199H and C199S forms indicated that these forms behave nearly identical to the WT enzyme. Furthermore, introduction of the C199H mutation in a truncated form of MutY (C199HT) allowed for crystallization and structural characterization of the modified [4Fe-4S] cluster coordination. The C199HT structure showed that histidine coordinated to the iron cluster although comparison to the structure of the WT truncated enzyme indicated that the occupancy of iron at the modified position had been reduced to 60%. Electron paramagnetic resonance (EPR) spectroscopy on samples of C199HT indicates that a significant percentage (15-30%) of iron clusters were of the [3Fe-4S]1+ form. Oxidation of the C199HT enzyme with ferricyanide increases the amount of the 3Fe cluster by approximately 2-fold. Detailed kinetic analysis on samples containing a mixture of [3Fe-4S]1+ and [4Fe-4S]2+ forms indicated that the reactivity of the [3Fe-4S]1+ C199HT enzyme does not differ significantly from that of the WT truncated enzyme. The relative resistance of the [4Fe-4S]2+ cluster toward oxidation, as well as the retention of activity of the [3Fe-4S]1+ form, may be an important aspect of the role of MutY in repair of DNA damage resulting from oxidative stress.
大肠杆菌DNA修复酶MutY在识别和修复DNA中的7,8 - 二氢 - 8 - 氧代 - 2'-脱氧鸟苷 - 2'-脱氧腺苷（OG*A）错配方面发挥着重要作用。MutY通过催化异常腺嘌呤的去糖基化作用，防止因OG对面错误掺入A而导致的DNA突变。MutY是DNA修复酶独特亚家族的代表，该亚家族还含有一个[4Fe - 4S]2+簇，其与底物识别有关。此前，我们利用定点诱变分别用丝氨酸、组氨酸或丙氨酸取代大肠杆菌MutY中与[4Fe - 4S]2+簇配位的半胱氨酸配体。这些实验表明，在MutY的199位，组氨酸与铁硫簇的配位可能是可行的。对C199H和C199S形式进行纯化和酶分析表明，这些形式的行为与野生型酶几乎相同。此外，在截短形式的MutY（C199HT）中引入C199H突变，使得能够对修饰后的[4Fe - 4S]簇配位进行结晶和结构表征。C199HT结构表明组氨酸与铁簇配位，尽管与野生型截短酶的结构比较表明修饰位置的铁占有率已降至60%。对C199HT样品进行电子顺磁共振（EPR）光谱分析表明，相当比例（15 - 30%）的铁簇为[3Fe - 4S]1+形式。用铁氰化物氧化C199HT酶会使3Fe簇的量增加约2倍。对含有[3Fe - 4S]1+和[4Fe - 4S]2+形式混合物的样品进行详细的动力学分析表明，[3Fe - 4S]1+ C199HT酶的反应活性与野生型截短酶没有显著差异。[4Fe - 4S]2+簇对氧化的相对抗性以及[3Fe - 4S]1+形式活性的保留，可能是MutY在修复氧化应激导致的DNA损伤中作用的一个重要方面。
