The Y430F mutant of Salmonella d-ornithine/d-lysine decarboxylase has altered stereospecificity and a putrescine allosteric activation site.
The Y430F mutant of Salmonella d-ornithine/d-lysine decarboxylase has altered stereospecificity and a putrescine allosteric activation site.
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DOI：
10.1016/j.abb.2022.109429
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发表时间：
2022-11-30
影响因子：
3.9
通讯作者：
中科院分区：
文献类型：
--
作者：
d-鸟氨酸/d-赖氨酸脱羧酶（DOKDC）的酪氨酸-430位于活性位点，并被认为是该酶的d-立体特异性的原因。制备了Y430F突变型肠沙门氏菌血清型鼠伤寒杆菌DOKDC，并对其对D-赖氨酸、l-赖氨酸和鸟氨酸的催化活性进行了评价。动力学结果表明，Y430F突变体对D-赖氨酸、l-赖氨酸和鸟氨酸具有可测量的脱羧酶活性，而野生型DOKDC则没有。光谱实验表明，这些氨基酸与吡哆醛-5′-磷酸结合形成外醛胺配合物，λmax = 425 nm。此外，我们还获得了Y430F DOKDC与HEPES、腐胺、d-鸟氨酸、d-赖氨酸和d-精氨酸结合的晶体结构。d-氨基酸在晶体中结合形成宝石二胺和外部醛二胺络合物的平衡混合物。此外，晶体结构揭示了一个意想不到的腐胺变构产物激活位点，位于两个活性位点之间的2倍轴上。腐胺通过在Asp-361和Gln-358的特异性螺旋上提供铵基的氢键结合。加入0.1-1 mM腐胺消除了稳态动力学的滞后，消除了s型动力学。利用AlphaFold2对催化环进行了建模，模型显示，Glu-181可以与结合的腐胺形成额外的氢键，可能稳定了催化闭合构象。
Tyrosine-430 of d-ornithine/d-lysine decarboxylase (DOKDC) is located in the active site, and was suggested to be responsible for the D-stereospecificity of the enzyme. We have prepared the Y430F mutant form of Salmonella enterica serovar typhimurium DOKDC and evaluated its catalytic activity with D- and l-lysine and ornithine. The kinetic results show that the Y430F mutant has measurable decarboxylase activity with both D- and l-lysine and ornithine, which wild type DOKDC does not. Spectroscopic experiments show that these amino acids bind to form external aldimine complexes with the pyridoxal-5′-phosphate with λmax = 425 nm. In addition, we have obtained crystal structures of Y430F DOKDC bound to HEPES, putrescine, d-ornithine, d-lysine, and d-arginine. The d-amino acids bind in the crystals to form equilibrium mixtures of gem-diamine and external aldimine complexes. Furthermore, the crystal structures reveal an unexpected allosteric product activator site for putrescine located on the 2-fold axis between the two active sites. Putrescine binds by donating hydrogen bonds from the ammonium groups to Asp-361 and Gln-358 in the specificity helix of both chains. Addition of 0.1–1 mM putrescine eliminates the lag in steady state kinetics and abolishes the sigmoid kinetics. The catalytic loop was modeled with AlphaFold2, and the model shows that Glu-181 can form additional hydrogen bonds with the bound putrescine, likely stabilizing the catalytic closed conformation.
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影响因子：
16.6
作者：
Bryant P;Pozzati G;Elofsson A
通讯作者：
Elofsson A
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
3.9
作者：
Maly, Martin;Diederichs, Kay;Kolenko, Petr
通讯作者：
Kolenko, Petr
影响因子：
2.9
作者：
Jackson, LK;Brooks, HB;Phillips, MA
通讯作者：
Phillips, MA
影响因子：
30.3
作者：
Kroeger, Carsten;Colgan, Aoife;Hinton, Jay C. D.
通讯作者：
Hinton, Jay C. D.