Stapon Anthony, Li Rongfeng, Townsend Craig A
Department of Chemistry, The Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, USA.
J Am Chem Soc. 2003 Jul 16;125(28):8486-93. doi: 10.1021/ja034248a.
(5R)-Carbapen-2-em-3-carboxylic acid is the simplest structurally among the naturally occurring carbapenem beta-lactam antibiotics. It co-occurs with two saturated (3S,5S)- and (3S,5R)-carbapenam carboxylic acids. Confusion persists in the literature about the signs of rotation and absolute configurations of these compounds that is resolved in this paper. (3S,5S)-Carbapenam carboxylic acid was prepared from L-pyroglutamic acid to unambiguously establish its absolute configuration as identical to the natural product isolated from Serratia marcescens and from overexpression of the biosynthetic genes carAB in Escherichia coli. L-Proline labeled with deuterium or tritium at the diastereotopic C-5 methylene loci was shown to incorporate one label at the bridgehead of (3S,5S)-carbapenam carboxylic acid, but not into the "inverted" (3S,5R)-carbapenam carboxylic acid or the final carbapenem product. CarC, the third enzyme of the biosynthetic pathway required to synthesize the carbapenem, was demonstrated in cell-free studies to be dependent on alpha-ketoglutarate and ascorbate in keeping with weak sequence identities with other non-heme iron, alpha-ketoglutarate-dependent oxygenases. CarC mediated the stereoinversion of synthetic (3S,5S)-carbapenam carboxylic acid to the (5R)-carbapenem as judged by bioassay. These findings suggest that L-proline is desaturated to pyrroline-5-carboxylic acid prior to uptake into the biosynthetic pathway. The loss of the bridgehead hydrogen from the (3S,5S)-carbapenam during the ring inversion process to form the epimeric (3S,5R)-carbapenam and desaturation to the (5R)-carbapenem are proposed to be coupled by CarC to the reduction of dioxygen to drive the formation of these higher energy products, an unprecedented reaction for this enzyme class.
(5R)-碳青霉烯-2-烯-3-羧酸是天然存在的碳青霉烯β-内酰胺抗生素中结构最简单的。它与两种饱和的(3S,5S)-和(3S,5R)-碳青霉烷羧酸共存。文献中对于这些化合物的旋光符号和绝对构型仍存在混淆，本文对此进行了澄清。(3S,5S)-碳青霉烷羧酸由L-焦谷氨酸制备，以明确确定其绝对构型与从粘质沙雷氏菌分离的天然产物以及在大肠杆菌中生物合成基因carAB的过表达产物相同。在非对映异位的C-5亚甲基位点用氘或氚标记的L-脯氨酸显示，在(3S,5S)-碳青霉烷羧酸的桥头掺入一个标记，但不掺入“反转的”(3S,5R)-碳青霉烷羧酸或最终的碳青霉烯产物。CarC是合成碳青霉烯所需生物合成途径的第三种酶，在无细胞研究中表明，它依赖于α-酮戊二酸和抗坏血酸，这与其他非血红素铁、α-酮戊二酸依赖性加氧酶的弱序列同一性一致。通过生物测定判断，CarC介导了合成的(3S,5S)-碳青霉烷羧酸向(5R)-碳青霉烯的立体反转。这些发现表明，L-脯氨酸在被摄取到生物合成途径之前先脱饱和为吡咯啉-5-羧酸。在环反转过程中，(3S,5S)-碳青霉烷桥头氢的丢失形成差向异构的(3S,5R)-碳青霉烷并脱饱和为(5R)-碳青霉烯，推测是由CarC与双氧的还原偶联，以驱动这些高能产物的形成，这是该酶类前所未有的反应。
