Structural and mutational studies on substrate specificity and catalysis of Salmonella typhimurium D-cysteine desulfhydrase.
Structural and mutational studies on substrate specificity and catalysis of Salmonella typhimurium D-cysteine desulfhydrase.
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DOI：
10.1371/journal.pone.0036267
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Murthy MR
中科院分区：
文献类型：
--
作者：
Bharath SR;Bisht S;Harijan RK;Savithri HS;Murthy MR
鼠伤寒沙门氏菌DCyD（StDCyD）是一种折叠II型吡哆醛5′磷酸（PLP）依赖性酶，催化D-Cys降解为H2S和丙酮酸。它还能有效降解β-氯-D-丙氨酸（βCDA）。D-丝氨酸是一种差的底物，而该酶对L-丝氨酸和1-氨基-1-羧基环丙烷（ACC）是无活性的。本文报道了StDCyD和在D-Cys、βCDA、ACC、D-Ser、L-Ser、D-环丝氨酸（DCS）和L-环丝氨酸（LCS）存在下获得的晶体的X射线晶体结构，分辨率范围为1.7 - 2.6 μ m。由小结构域（残基48-161）和大结构域（残基1-47和162-328）组成的StDCyD的多肽折叠类似于其他折叠II型PLP依赖性酶。在D-Cys和βCDA存在下获得的结构显示产物丙酮酸结合在距离活性位点4.0-6.0 nm的位点处。ACC形成外部醛亚胺复合物，而D-和L-Ser非共价结合，表明与这些配体的反应分别在Cα质子提取和transimination步骤中被阻止。在与DCS或LCS共结晶的StDCyD的活性位点中，观察到磷酸吡哆胺（PMP）的电子密度。浸泡在含有这些配体的混合物中的晶体显示出PLP-环丝氨酸的密度。光谱观察也表明PMP的形成环丝氨酸的水解。突变研究表明，Ser 78和Gln 77是酶特异性的关键决定因素，Tyr 287的酚盐负责从D-Cys提取Cα质子。基于这些研究，提出了StDCyD降解D-Cys的可能机制。
Salmonella typhimurium DCyD (StDCyD) is a fold type II pyridoxal 5′ phosphate (PLP)-dependent enzyme that catalyzes the degradation of D-Cys to H2S and pyruvate. It also efficiently degrades β-chloro-D-alanine (βCDA). D-Ser is a poor substrate while the enzyme is inactive with respect to L-Ser and 1-amino-1-carboxy cyclopropane (ACC). Here, we report the X-ray crystal structures of StDCyD and of crystals obtained in the presence of D-Cys, βCDA, ACC, D-Ser, L-Ser, D-cycloserine (DCS) and L-cycloserine (LCS) at resolutions ranging from 1.7 to 2.6 Å. The polypeptide fold of StDCyD consisting of a small domain (residues 48–161) and a large domain (residues 1–47 and 162–328) resembles other fold type II PLP dependent enzymes. The structures obtained in the presence of D-Cys and βCDA show the product, pyruvate, bound at a site 4.0–6.0 Å away from the active site. ACC forms an external aldimine complex while D- and L-Ser bind non-covalently suggesting that the reaction with these ligands is arrested at Cα proton abstraction and transimination steps, respectively. In the active site of StDCyD cocrystallized with DCS or LCS, electron density for a pyridoxamine phosphate (PMP) was observed. Crystals soaked in cocktail containing these ligands show density for PLP-cycloserine. Spectroscopic observations also suggest formation of PMP by the hydrolysis of cycloserines. Mutational studies suggest that Ser78 and Gln77 are key determinants of enzyme specificity and the phenolate of Tyr287 is responsible for Cα proton abstraction from D-Cys. Based on these studies, a probable mechanism for the degradation of D-Cys by StDCyD is proposed.
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影响因子：
5.4
作者：
Bharath, Sakshibeedu Rajegowda;Bisht, Shveta;Murthy, Mattur Ramabhadrashastry Narasimha
通讯作者：
Murthy, Mattur Ramabhadrashastry Narasimha
影响因子：
4.1
作者：
Ito, Tomokazu;Hemmi, Hisashi;Yoshimura, Tohru
通讯作者：
Yoshimura, Tohru
影响因子：
3.3
作者：
INOUYE, S;TSURUOKA, T;MATSUHASHI, M
通讯作者：
MATSUHASHI, M
影响因子：
6.1
作者：
Cohen, GH
通讯作者：
Cohen, GH
DOI：
10.1002/prot.10286
发表时间：
2003-02-15
期刊：
PROTEINS-STRUCTURE FUNCTION AND GENETICS
影响因子：
--
作者：
Lovell, SC;Davis, IW;Richardson, DC
通讯作者：
Richardson, DC