Enzyme functional evolution through improved catalysis of ancestrally nonpreferred substrates

Abstract

In this study, we investigated the role for ancestral functional variation that may be selected upon to generate protein functional shifts using ancestral protein resurrection, statistical tests for positive selection, forward and reverse evolutionary genetics, and enzyme functional assays. Data are presented for three instances of protein functional change in the salicylic acid/benzoic acid/theobromine (SABATH) lineage of plant secondary metabolite-producing enzymes. In each case, we demonstrate that ancestral nonpreferred activities were improved upon in a daughter enzyme after gene duplication, and that these functional shifts were likely coincident with positive selection. Both forward and reverse mutagenesis studies validate the impact of one or a few sites toward increasing activity with ancestrally nonpreferred substrates. In one case, we document the occurrence of an evolutionary reversal of an active site residue that reversed enzyme properties. Furthermore, these studies show that functionally important amino acid replacements result in substrate discrimination as reflected in evolutionary changes in the specificity constant (k cat /K M ) for competing substrates, even though adaptive substitutions may affect K M and k cat separately. In total, these results indicate that nonpreferred, or even latent, ancestral protein activities may be coopted at later times to become the primary or preferred protein activities.carboxyl methyltransferase | adaptive protein evolution

Convergent evolution of caffeine in plants by co-option of exapted ancestral enzymes

“…The existence of exapted ancestral enzymes such as CisAncXMT2 resolves one of the fundamental problems of the cumulative hypothesis because multiple steps of a pathway could evolve simultaneously, thereby avoiding the need to assume the existence of selectively advantageous intermediates. These results also point to a crucial role for ancestral promiscuous activities postulated as part of the patchwork hypothesis and protein engineering studies (31) and reported previously for other SABATH enzymes (46).…”

“…Ancestral O-methylation of the carboxyl moiety of benzoic and salicylic acid might have promoted the evolution of N-methylation of xanthine alkaloids because of common attributes of the active sites that would need to accommodate the largely planar rings of both classes of substrates. Indeed, a paralogous SABATH methyltransferase specialized for methylation of the carboxyl group of nicotinic acid (which is also an N-heterocyclic substrate) also recently arose from ancestral enzymes that exhibited activities with benzoic and salicylic acid (46). Although these data indicate that ancestral activity with benzoic and salicylic acid in the XMT lineage allowed for subsequent co-option of the descendant enzymes to form caffeine, how recruitment of the enzymes into a functional pathway occurred remains unknown.…”

“…S9). This residue is likely part of the active site and known to control substrate preference in other SABATH enzymes (46). Experimental mutagenesis of His150 to Asn in CisAncXMT2 (Fig.…”

Whole-genome duplication in teleost fishes and its evolutionary consequences

“…Physical modeling of amino acid chains suggests that EAC preferentially takes place under moderate selective pressure and therefore likely in genes that are not essential for survival, but that can substantially improve fitness if optimized (Sikosek et al, 2012). So far, duplicate gene evolution by EAC has been mainly a theoretical model and only few cases indicating EAC have been documented (DesMarais and Rausher, 2008;Deng et al, 2010;Huang et al, 2012). One reason for the scarcity of examples is related to the difficulty 13 to ascertain whether the criteria for EAC are fulfilled, in particular whether functions were improved compared to the ancestral gene and whether this improvement was really constrained before duplication (Barkman and Zhang, 2009).…”

Network-level architecture and the evolutionary potential of underground metabolism

“…Central to this "patchwork" model of pathway evolution is the notion that many enzymes have limited substrate specificities and can catalyze, albeit at low rates, reactions other than those for which they have evolved (also referred to as enzyme promiscuity) (4). These so-called underground (5) or side activities are prevalent (6)(7)(8) and were shown to serve as starting points for the evolution of novel functions both in directed evolution experiments (9) and in the diversification of gene families in the wild (7). However, how the underground catalytic repertoire encoded in the genome can generate novelties within the context of the existing metabolic network remains unknown.…”