Multicopy suppression underpins metabolic evolvability

Abstract

Our understanding of the origins of new metabolic functions is based upon anecdotal genetic and biochemical evidence. Some auxotrophies can be suppressed by overexpressing substrate-ambiguous enzymes (i.e., those that catalyze the same chemical transformation on different substrates). Other enzymes exhibit weak but detectable catalytic promiscuity in vitro (i.e., they catalyze different transformations on similar substrates). Cells adapt to novel environments through the evolution of these secondary activities, but neither their chemical natures nor their frequencies of occurrence have been characterized en bloc. Here, we systematically identified multifunctional genes within the Escherichia coli genome. We screened 104 single-gene knockout strains and discovered that many (20%) of these auxotrophs were rescued by the overexpression of at least one noncognate E. coli gene. The deleted gene and its suppressor were generally unrelated, suggesting that promiscuity is a product of contingency. This genome-wide survey demonstrates that multifunctional genes are common and illustrates the mechanistic diversity by which their products enhance metabolic robustness and evolvability.

Fig. 1

Phosphatase substrate ambiguity. SerB is the phosphoserine phosphatase that catalyzes the final step in the biosynthesis of serine from 3-phosphoglycerate (top). Deletion of serB can be rescued by expression of 2 other HAD-like phosphatases: Gph, which hydrolyzes the phosphate monoester 2-phosphoglycolate (middle), and HisB, which ordinarily catalyzes the phosphorolysis of histidinol phosphate (bottom).

Fig. 2

Catalytic promiscuity among chorismate-utilizing enzymes. PabB catalyzes the formation of 4-amino-4-deoxychorismate; the ΔpabB strain can be rescued by expression of its homologue, MenF (isochorismate synthase). The 2 enzymes act via a common intermediate, generated in an S_N2′ mechanism that leads to addition of a nucleophile at C2, with concomitant elimination of the C4 hydroxyl group (He et al. 2004). In PabB, the nucleophile is the ∊-amino group of an active site lysine; the result is a covalent intermediate. In MenF, the nucleophile is assumed to be water, although its isozyme, EntC, can also utilize ammonia. Our results suggest that product release by MenF is sufficiently slow that ammonia can enter the active site and effect a second S_N2′ displacement, yielding aminodeoxychorismate in quantities that enable cell survival in the absence of PabB.