Wang Yu, Jones Michael K, Xu Huimin, Ray W Keith, White Robert H
Biochemistry. 2015 May 19;54(19):2997-3008. doi: 10.1021/acs.biochem.5b00176.
A single enzyme, 4-(hydroxymethyl)-2-furancarboxaldehyde-phosphate synthase (MfnB), from the methanogen Methanocaldococcus jannaschii catalyzed at least 10 separate chemical reactions in converting two molecules of glyceraldehyde-3-P (GA-3-P) to 4-(hydroxymethyl)-2-furancarboxaldehyde-P (4-HFC-P), the first discrete intermediate in the biosynthetic pathway to the furan moiety of the coenzyme methanofuran. Here we describe the biochemical characterization of the recombinantly expressed MfnB to understand its catalytic mechanism. Site-directed mutagenesis showed that the strictly conserved residues (Asp25, Lys27, Lys85, and Asp151) around the active site are all essential for enzyme catalysis. Matrix-assisted laser desorption/ionization analysis of peptide fragments of MfnB incubated with GA-3-P followed by NaBH₄ reduction and trypsin digestion identified a peptide with a mass/charge ratio of 1668.8 m/z present only in the D25N, D151N, and K155R mutants, which is consistent with Lys27 having increased by a mass of 58 m/z, indicating that Lys27 forms a Schiff base with a methylglyoxal-like intermediate. In addition, incubation of MfnB with GA-3-P in the presence of deuterated water or incubation of MfnB with C-2 deuterated GA-3-P showed essentially no deuterium incorporated into the 4-HFC-P. Combined with structural analysis and molecular docking, we predict the potential binding sites for two GA-3P molecules in the active site. On the basis of our observations, a possible catalytic mechanism of MfnB is proposed in this study. A phosphate elimination reaction and a triose phosphate isomerase-like reaction occur at the GA-3-P binding site I and II, respectively, prior to the aldol condensation between the enzyme-bound enol form of methylglyoxal and dihydroxyacetone phosphate (DHAP), after which the catalytic cycle is completed by a cyclization and two dehydration reactions assisted by several general acids/bases at the same active site.
来自嗜热甲烷球菌的单一酶，即4-(羟甲基)-2-呋喃甲醛磷酸合酶（MfnB），在将两分子甘油醛-3-磷酸（GA-3-P）转化为4-(羟甲基)-2-呋喃甲醛磷酸（4-HFC-P）的过程中催化至少10个不同的化学反应，4-HFC-P是辅酶甲烷呋喃呋喃部分生物合成途径中的第一个离散中间体。在此，我们描述了重组表达的MfnB的生化特性，以了解其催化机制。定点诱变表明，活性位点周围严格保守的残基（Asp25、Lys27、Lys85和Asp151）对酶催化都是必不可少的。对与GA-3-P孵育、然后用NaBH₄还原并经胰蛋白酶消化的MfnB肽片段进行基质辅助激光解吸/电离分析，鉴定出仅在D25N、D151N和K155R突变体中存在的质荷比为1668.8 m/z的肽段，这与Lys27质量增加58 m/z一致，表明Lys27与甲基乙二醛样中间体形成了席夫碱。此外，在氘代水存在下将MfnB与GA-3-P孵育，或将MfnB与C-2氘代GA-3-P孵育，结果显示基本上没有氘掺入4-HFC-P中。结合结构分析和分子对接，我们预测了活性位点中两个GA-3P分子的潜在结合位点。基于我们的观察结果，本研究提出了MfnB可能的催化机制。在酶结合的甲基乙二醛烯醇形式与磷酸二羟丙酮（DHAP）之间进行羟醛缩合之前，GA-3-P结合位点I和II分别发生磷酸消除反应和磷酸丙糖异构酶样反应，之后通过同一活性位点的几个通用酸/碱辅助的环化反应和两个脱水反应完成催化循环。
