Biosynthesis of pyrrolizidine alkaloids: putrescine and spermidine are essential substrates of enzymatic homospermidine formation

Abstract

sym-Homospermidine, the first pathway-specific intermediate of pyrrolizidine alkaloid (PA) biosynthesis, is formed from two moles of putrescine in an NAD+-dependent reaction catalyzed by homospermidine synthase (HSS). In the overall process catalyzed by HSS, NAD+ seems to function as hydride acceptor in the first part of the reaction and subsequently as hydride donor in the second part. This was proved with chirally Cl-deuterated putrescines. Both a bacterial HSS (isolated from Rhodopseudomonas viridis) and the enzyme from PA-producing Eupatorium cannabinum transformed (R)-[1-2H]putrescine and (S)-[1-2H]-putrescine into homospermidine with a 100% retention of deuterium. Studies with deuterated and 14C-labeled substrates revealed that in the presence of putrescine spermidine is a substrate of HSS. The putrescine semialdehyde moiety of spermidine is combined with putrescine and 1,3-diaminopropane is released. Kinetic studies with plant HSS documented that the putrescine semialdehyde moiety of spermidine is incorporated into homospermidine with the same affinity (Km) and activity (Vmax) as putrescine. Plant HSS is highly specific for putrescine and spermidine; spermidine is not a substrate in the absence of putrescine. It is suggested that in the biosynthesis of homospermidine and thus PAs, one of the two C4 units is derived from spermidine and the other one from putrescine. A scheme of the HSS-catalyzed reaction is proposed that fully accounts for the enzymatic data and the results of previous tracer studies. Bacterial HSS is less substrate specific and not as well adapted as plant HSS to accept spermidine as a substrate.