A single mutation in the active site swaps the substrate specificity of N-acetyl-L-ornithine transcarbamylase and N-succinyl-L-ornithine transcarbamylase
A single mutation in the active site swaps the substrate specificity of N-acetyl-L-ornithine transcarbamylase and N-succinyl-L-ornithine transcarbamylase
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DOI：
10.1110/ps.072919907
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发表时间：
2007-08-01
期刊：
影响因子：
8
通讯作者：
Tuchman, Mendel
中科院分区：
文献类型：
--
作者：
Shi, Dashuang;Yu, Xiaolin;Tuchman, Mendel
转氨甲酰酶催化氨甲酰基团从氨甲酰磷酸（CP）转移到第二底物如天冬氨酸、鸟氨酸或腐胺的氨基基团。以前，来自野油菜黄单胞菌的转氨甲酰酶的结构测定导致发现新的N-乙酰鸟氨酸转氨甲酰酶（AOTCase），其催化N-乙酰鸟氨酸的氨甲酰化。最近，一种新的N-琥珀酰鸟氨酸转氨甲酰酶（SOTCase）从脆弱拟杆菌被确定。从X. campestris和SOTCase来自B. fragilis分析表明，Glu 92（X. campestris编号）在区分AOTCase和SOTCase中起关键作用。AOTCase的E92 P、E92 S、E92 V和E92 A突变体的酶促测定证明，这些突变中的每一个都将AOTCase转化为SOTCase。类似地，B. fragilis SOTCase（相当于X. campestris AOTCase）将SOTCase转换为AOTCase。因此，单个氨基酸取代足以交换AOTCase和SOTCase的底物特异性。这些突变体与CP和N-乙酰基-L-正缬氨酸（N-乙酰基-L-鸟氨酸的类似物）或N-琥珀酰-L-正缬氨酸（N-琥珀酰-L-鸟氨酸的类似物）复合的X射线晶体结构证实了这种转化。除了Glu 92（X. campestris编号），AOTCase中的其他残基如Asn 185和Lys 30，它们通过桥接水分子参与结合底物，有助于确定AOTCase的底物特异性。这些结果为一组被错误地注释为鸟氨酸转氨甲酰酶的转氨甲酰酶样蛋白提供了正确的注释（AOTCase或SOTCase）（OTCase，EC 2.1.3.3）.
Transcarbamylases catalyze the transfer of the carbamyl group from carbamyl phosphate (CP) to an amino group of a second substrate such as aspartate, ornithine, or putrescine. Previously, structural determination of a transcarbamylase from Xanthomonas campestris led to the discovery of a novel N-acetylornithine transcarbamylase (AOTCase) that catalyzes the carbamylation of N-acetylornithine. Recently, a novel N-succinylornithine transcarbamylase (SOTCase) from Bacteroides fragilis was identified. Structural comparisons of AOTCase from X. campestris and SOTCase from B. fragilis revealed that residue Glu92 (X. campestris numbering) plays a critical role in distinguishing AOTCase from SOTCase. Enzymatic assays of E92P, E92S, E92V, and E92A mutants of AOTCase demonstrate that each of these mutations converts the AOTCase to an SOTCase. Similarly, the P90E mutation in B. fragilis SOTCase (equivalent to E92 in X. campestris AOTCase) converts the SOTCase to AOTCase. Hence, a single amino acid substitution is sufficient to swap the substrate specificities of AOTCase and SOTCase. X- ray crystal structures of these mutants in complexes with CP and N-acetyl-L-norvaline (an analog of N-acetyl-L-ornithine) or N-succinyl-L- norvaline (an analog of N-succinyl-L-ornithine) substantiate this conversion. In addition to Glu92 (X. campestris numbering), other residues such as Asn185 and Lys30 in AOTCase, which are involved in binding substrates through bridging water molecules, help to define the substrate specificity of AOTCase. These results provide the correct annotation (AOTCase or SOTCase) for a set of the transcarbamylase- like proteins that have been erroneously annotated as ornithine transcarbamylase (OTCase, EC 2.1.3.3).