Heme is an essential prosthetic group for many proteins involved in fundamental biological processes in all three domains of life. In Eukaryota and Bacteria heme is formed via a conserved and well-studied biosynthetic pathway. Surprisingly, in Archaea heme biosynthesis proceeds via an alternative route which is poorly understood. In order to formulate a working hypothesis for this novel pathway, we searched 59 completely sequenced archaeal genomes for the presence of gene clusters consisting of established heme biosynthetic genes and colocalized conserved candidate genes. Within the majority of archaeal genomes it was possible to identify such heme biosynthesis gene clusters. From this analysis we have been able to identify several novel heme biosynthesis genes that are restricted to archaea. Intriguingly, several of the encoded proteins display similarity to enzymes involved in heme d1 biosynthesis. To initiate an experimental verification of our proposals two Methanosarcina barkeri proteins predicted to catalyze the initial steps of archaeal heme biosynthesis were recombinantly produced, purified, and their predicted enzymatic functions verified. 1. Introduction Heme, a modified tetrapyrrole, acts as an essential prosthetic group in many enzymes, sensory, and regulatory proteins. Hemes are also essential components of electron transport chains driving aerobic and anaerobic respiration and photosynthesis in almost all living organisms. Consequently, heme-containing proteins are found in all three domains of life, the Eukaryota, the Bacteria, and the Archaea. The biosynthesis of this important and ubiquitously distributed molecule has been intensively studied in eukaryotic and bacterial organisms, but little is known about heme biosynthesis in archaea. It is now well established for bacteria and eukarya that heme biosynthesis proceeds along a conserved pathway with highly related enzymes and identical biosynthetic intermediates (Figure 1(a)) [1]. Heme synthesis represents just one component of a larger, branched tetrapyrrole biosynthesis pathway, which is also responsible for the synthesis of chlorophylls, bacteriochlorophylls, cobalamin, siroheme, heme d1 and coenzyme F430 (Figure 1(b)) [2]. Figure 1: Tetrapyrrole biosynthesis pathways. (a) Heme biosynthesis in most bacteria and the Eukaryota. The first common precursor in the classical heme biosynthesis pathway is ALA of which eight molecules are converted into UROGEN in three consecutive enzymatic steps. UROGEN is then further converted into heme through successive modifications of the macrocycle
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