Metabolites: a helping hand for pathway evolution? Steffen Schmidt 1,2,5 , Shamil Sunyaev 1,3 , Peer Bork 1,5 and Thomas Dandekar 1,4,5 1 European Molecular Biology Laboratory Heidelberg, Postfach 102209, D-69012 Heidelberg, Germany 2 University of Heidelberg, Department of Parasitology, INF 324, D-69120 Heidelberg, Germany 3 Present address: Genetics Division, Department of Medicine, Brigham & Women’s Hospital and Harvard Medical School, 20 Shattuck Str. Thorn 10, Boston MA 02115, USA 4 University of Wu ¨ rzburg, Department of Bioinformatics, Biozentrum, Am Hubland, D-97074 Wu ¨ rzburg, Germany 5 Max Delbru ¨ ck-Centrum fu ¨ r Molekulare Medizin, Robert-Ro ¨ ssle-Str. 10, D-13122 Berlin-Buch, Germany The evolution of enzymes and pathways is under debate. Recent studies show that recruitment of single enzymes from different pathways could be the driving force for pathway evolution. Other mechanisms of evol- ution, such as pathway duplication, enzyme specializ- ation, de novo invention of pathways or retro-evolution of pathways, appear to be less abundant. Twenty per- cent of enzyme superfamilies are quite variable, not only in changing reaction chemistry or metabolite type but in changing both at the same time. These variable superfamilies account for nearly half of all known reac- tions. The most frequently occurring metabolites pro- vide a helping hand for such changes because they can be accommodated by many enzyme superfamilies. Thus, a picture is emerging in which new pathways are evolving from central metabolites by preference, thereby keeping the overall topology of the metabolic network. It is well known that enzymes are specific and that they catalyze individual reactions with surprising accuracy and speed. However, for adaptation and evolution the opposite is required: new substrates must be recognized and new enzyme activities evolved. But where does the flexibility and plasticity of metabolic pathways and new enzyme activities come from? The majority of recent studies have concentrated on the level of pathways or enzymes as well as the variability of enzymes in their reaction chemistry [1–14]. However, the variability in substrates and products – metabolites – must also be considered. Pathway evolution theories On the level of pathway evolution, several hypotheses have been proposed (Fig. 1). First, pathways might have evolved spontaneously without adopting existing enzymes (Fig. 1a). For example, different tRNA synthetases seem to have initially evolved independently and then later have become involved in different pathways such as protein translation, tRNA dependent transamidation and non- discriminating acylation [15]. Second, the hypothesis of ‘retro-evolution’ of pathways [16,17] proposes that the selective pressure on a pathway mainly targets the successful production of its end-product (Fig. 1b). The formation of the required end-product from an intermedi- ate metabolite increases the fitness of the organism. As the end-product can be derived from more and more ‘distant’ metabolites, fitness increases and the pathway evolves backwards. This retro-evolution has been proposed for both the glycolytic [18] and the mandelate pathway [19]. Third, pathways might have evolved from multifunctional enzymes [20] (Fig. 1c). Starting from a multifunctional enzyme catalyzing consecutive steps, the pathway might have then evolved by duplication and diversification of this precursor enzyme to the more specific and efficient enzymes known today, which catalyze only one step each in the pathway. O’Brien and Herschlag [21] analyzed several enzymes with alternative reactions distinct from their normal biological reaction to support the concept that broader substrates and reaction specificities are sub- sequently captured by adaptive evolution. Existing multi- functional enzymes, such as the carbamoyl phosphate synthase, are already used in diverse functions and pathways, such as b-D-glucan hydrolases in higher plants, and might be precursors to new pathways [6]. This hypothesis assumes that a single enzyme becomes specialized, but there is also the possibility that whole pathways (as a unit) became duplicated and diverted (Fig. 1d). This mechanism of acquiring new function has been examined for a long time [22] and can be readily identified using comparative genomics [10,23]. Examples include tryptophan and histidine biosynthesis [24,25]; these two pathways consist of several steps that have similar reaction chemistry and that are catalyzed by homologous enzymes – probably the result of early pathway duplication. Finally, pathways might have evolved by ‘recruiting’ enzymes from existing pathways, resulting in a mosaic or ‘patchwork’ of homologous enzymes that catalyze reactions in distinct pathways [25–27] (Fig. 1e). Observations indicate that one type of enzyme fold (e.g. TIM barrel [4]) or one enzyme super- family [7] might catalyze similar reactions, but occur in different pathways owing to widespread recruitment. Such versatility has been found in many Escherichia coli small molecule metabolism enzymes [12,13]. Corresponding author: Thomas Dandekar (Dandekar@EMBL.de). Review TRENDS in Biochemical Sciences Vol.28 No.6 June 2003 336 http://tibs.trends.com 0968-0004/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0968-0004(03)00114-2