Harijan Rajesh K, Kiema Tiila-Riikka, Syed Shahan M, Qadir Imran, Mazet Muriel, Bringaud Frédéric, Michels Paul A M, Wierenga Rik K
Faculty of Biochemistry and Molecular Medicine, Biocenter Oulu, University of Oulu, FIN-90014, Finland.
Present address: Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Protein Eng Des Sel. 2017 Mar 1;30(3):225-233. doi: 10.1093/protein/gzw080.
Structures of the C123A variant of the dimeric Leishmania mexicana SCP2-thiolase (type-2) (Lm-thiolase), complexed with acetyl-CoA and acetoacetyl-CoA, respectively, are reported. The catalytic site of thiolase contains two oxyanion holes, OAH1 and OAH2, which are important for catalysis. The two structures reveal for the first time the hydrogen bond interactions of the CoA-thioester oxygen atom of the substrate with the hydrogen bond donors of OAH1 of a CHH-thiolase. The amino acid sequence fingerprints ( xS, EAF, G P) of three catalytic loops identify the active site geometry of the well-studied CNH-thiolases, whereas SCP2-thiolases (type-1, type-2) are classified as CHH-thiolases, having as corresponding fingerprints xS, DCF and G P. In all thiolases, OAH2 is formed by the main chain NH groups of two catalytic loops. In the well-studied CNH-thiolases, OAH1 is formed by a water (of the Wat-Asn(NEAF) dyad) and NE2 (of the GHP-histidine). In the two described liganded Lm-thiolase structures, it is seen that in this CHH-thiolase, OAH1 is formed by NE2 of His338 (HDCF) and His388 (GHP). Analysis of the OAH1 hydrogen bond networks suggests that the GHP-histidine is doubly protonated and positively charged in these complexes, whereas the HDCF histidine is neutral and singly protonated.
报道了二聚体墨西哥利什曼原虫SCP2 - 硫解酶（2型）（Lm - 硫解酶）的C123A变体分别与乙酰辅酶A和乙酰乙酰辅酶A形成的复合物结构。硫解酶的催化位点包含两个氧负离子洞，即OAH1和OAH2，它们对催化作用很重要。这两种结构首次揭示了底物的辅酶A - 硫酯氧原子与CHH - 硫解酶OAH1的氢键供体之间的氢键相互作用。三个催化环的氨基酸序列指纹（xS、EAF、G P）确定了研究充分的CNH - 硫解酶的活性位点几何结构，而SCP2 - 硫解酶（1型、2型）被归类为CHH - 硫解酶，其相应指纹为xS、DCF和G P。在所有硫解酶中，OAH2由两个催化环的主链NH基团形成。在研究充分的CNH - 硫解酶中，OAH1由（Wat - Asn(NEAF)二元组中的）一个水分子和（GHP - 组氨酸的）NE2形成。在所述的两种配体结合的Lm - 硫解酶结构中，可以看到在这种CHH - 硫解酶中，OAH1由His338（HDCF）的NE2和His388（GHP）形成。对OAH1氢键网络的分析表明，在这些复合物中GHP - 组氨酸被双重质子化且带正电荷，而HDCF组氨酸是中性的且单质子化。
