Bansal Paramjit S, Torres Allan M, Crossett Ben, Wong Karen K Y, Koh Jennifer M S, Geraghty Dominic P, Vandenberg Jamie I, Kuchel Philip W
School of Molecular and Microbial Biosciences, University of Sydney, New South Wales 2006, Australia.
J Biol Chem. 2008 Apr 4;283(14):8969-75. doi: 10.1074/jbc.M709762200. Epub 2007 Dec 24.
The L-to-D-peptide isomerase from the venom of the platypus (Ornithorhyncus anatinus) is the first such enzyme to be reported for a mammal. In delineating its catalytic mechanism and broader roles in the animal, its substrate specificity was explored. We used N-terminal segments of defensin-like peptides DLP-2 and DLP-4 and natriuretic peptide OvCNP from the venom as substrates. The DLP analogues IMFsrs and ImFsrs (srs is a solubilizing chain; lowercase letters denote D-amino acid) were effective substrates for the isomerase; it appears to recognize the N-terminal tripeptide sequence Ile-Xaa-Phe-. A suite of 26 mutants of these hexapeptides was synthesized by replacing the second residue (Met) with another amino acid, viz. Ala, alpha-aminobutyric acid, Ile, Leu, Lys, norleucine, Phe, Tyr, and Val. It was shown that mutant peptides incorporating norleucine and Phe are substrates and exhibit L- or D-amino acid isomerization, but mutant peptides that contain residues with shorter, beta-branched or long side chains with polar terminal groups, viz. Ala, alpha-aminobutyric acid, Ile, Val, Leu, Lys, and Tyr, respectively, are not substrates. It was demonstrated that at least three N-terminal amino acid residues are absolutely essential for L-to-D-isomerization; furthermore, the third amino acid must be a Phe residue. None of the hexapeptides based on LLH, the first three residues of OvCNP, were substrates. A consistent 2-base mechanism is proposed for the isomerization; abstraction of a proton by 1 base is concomitant with delivery of a proton by the conjugate acid of a second base.
鸭嘴兽（Ornithorhyncus anatinus）毒液中的L-到D-肽异构酶是首次报道的来自哺乳动物的此类酶。在阐明其催化机制及其在动物体内的更广泛作用时，对其底物特异性进行了探索。我们使用了毒液中防御素样肽DLP-2和DLP-4的N端片段以及利钠肽OvCNP作为底物。DLP类似物IMFsrs和ImFsrs（srs是一个增溶链；小写字母表示D-氨基酸）是该异构酶的有效底物；它似乎识别N端三肽序列Ile-Xaa-Phe-。通过将第二个残基（Met）替换为另一种氨基酸，即丙氨酸、α-氨基丁酸、异亮氨酸、亮氨酸、赖氨酸、正亮氨酸、苯丙氨酸、酪氨酸和缬氨酸，合成了一组26种这些六肽的突变体。结果表明，包含正亮氨酸和苯丙氨酸的突变肽是底物，并表现出L-或D-氨基酸异构化，但分别含有短侧链、β-分支侧链或带有极性末端基团的长侧链残基的突变肽，即丙氨酸、α-氨基丁酸、异亮氨酸、缬氨酸、亮氨酸、赖氨酸和酪氨酸，不是底物。结果表明，至少三个N端氨基酸残基对于L到D异构化是绝对必需的；此外，第三个氨基酸必须是苯丙氨酸残基。基于OvCNP的前三个残基LLH的六肽都不是底物。提出了一种一致的双碱基异构化机制；一个碱基夺取质子与第二个碱基的共轭酸传递质子同时发生。
