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.

Fig. 1.

Relative enzyme activities of 13 SAMT, BSMT, and NAMT with 18 substrates arranged by evolutionary relationships. Hoya and Stephanotis are from Apocynaceae, whereas all other species are members of Solanaceae. Activity with the favored substrate was used to normalize all other activities for each enzyme, which range from 0 to 100. Most SAMT show high relative preference for salicylic acid (SA) relative to benzoic acid (BA) and all other substrates. Most BSMT show higher relative preference for BA than SA, although high activities are shown with o-anisic acid and anthranilic acid as well. NAMT exhibits highest preference for nicotinic acid (NA). The gene duplication event that gave rise to the entire lineage of enzymes shown is hypothesized to have occurred at node A (31) (Fig. S1). A gene duplication event at node D occurred early in Solanaceae evolutionary history and resulted in the SAMT and BSMT lineages of that family. A later duplication event is thought to have occurred at node E only within Nicotiana (30). Data for S. floribunda are from ref. . Numbered structures are as follow: 1, salicylic acid; 2, benzoic acid (BA); 3, 3-hydroxyBA; 4, 4-hydroxyBA; 5, 2,3-dihydroxyBA; 6, 2,4-dihydroxyBA; 7, 2,5-dihydroxyBA; 8, 2,6-dihydroxyBA; 9, 3,4-dihydroxyBA; 10, 3,5-dihydroxyBA; 11, cinnamic acid; 12, o-coumaric acid; 13, m-coumaric acid; 14, p-coumaric acid; 15, o-anisic acid; 16, anthranilic acid; 17, jasmonic acid; and 18, nicotinic acid.

Fig. 2.

Experimental results used to investigate the fate of ancestral nonpreferred enzyme activities after functional shifts. The tree shown is simplified from that shown in Fig. 1 with the same node labels. (A–G) Nodes for which relative enzyme preference for BA (green), SA (black), and NA (red) for either resurrected enzymes and their forward mutants or reverse mutants of modern-day enzymes (bold) was determined. Mean and SD are shown on the basis of at least two replicate assays. Color shown for lineages indicates the highest ancestral relative activity with a particular substrate. The ancestral enzyme at node A appears to have preferred BA but later evolved to prefer SA as shown for node B. This change in preference was concomitant with positive selection for the replacement of His by Met at position 201 (P < 0.05). The ancestral enzyme at node D had a 20-fold higher relative activity for SA over BA, but after gene duplication, the descendant enzyme at node E evolved a >20-fold higher relative activity for BA over SA. This evolutionary reversal appears to have been concomitant with positive selection for the reverse replacement of Met by His at position 201 (P < 0.05). The ancestral enzyme, ancMT-F, evolved high relative preference for NA from an ancestor that preferred BA (ancMT-E). Subsequently, preference for NA increased even more in NAMT, probably by the replacement of Phe420 by Tyr, although it is not clear whether positive selection was concomitant with this change (P > 0.05).