Overexpression of seven different genes restores growth of a ΔpdxB strain of E. coli, which cannot make pyridoxal phosphate (PLP), on M9/glucose.None of the enzymes encoded by these genes has a promiscuous 4-phosphoerythronate dehydrogenase activity that can replace the activity of PdxB.Overexpression of these genes restores PLP synthesis by three different serendipitous pathways that feed into the normal PLP synthesis pathway downstream of the blocked step.Reactions in one of these pathways are catalyzed by low-level activities of enzymes of unknown function and a promiscuous activity of an enzyme that normally has a role in another pathway; one reaction appears to be non-enzymatic.
Most metabolic enzymes are prodigious catalysts that have evolved to accelerate chemical reactions with high efficiency and specificity. However, many enzymes have inefficient promiscuous activities, as well, as a result of the assemblage of highly reactive catalytic residues and cofactors in active sites. Although promiscuous activities are generally orders of magnitude less efficient than well-evolved activities (O'Brien and Herschlag, 1998, 2001; Wang et al, 2003; Taylor Ringia et al, 2004), they often enhance reaction rates by orders of magnitude relative to those of uncatalyzed reactions (O'Brien and Herschlag, 1998, 2001). Thus, promiscuous activities provide a reservoir of novel catalytic activities that can be recruited to serve new functions.
The evolutionary potential of promiscuous enzymes extends beyond the recruitment of single enzymes to serve new functions. Microbes contain hundreds of enzymes—E. coli contains about 1700 (Freilich et al, 2005)—raising the possibility that promiscuous enzymes can be patched together to generate ‘serendipitous' pathways that are not part of normal metabolism. We distinguish serendipitous pathways from latent or cryptic pathways, which are bona fide pathways involving dedicated enzymes that are produced only under particular environmental circumstances. In contrast, serendipitous pathways are patched together from enzymes that normally serve other functions and are not regulated in a coordinated manner in response to the need to synthesize or degrade a metabolite.
In this study, we describe the discovery of three serendipitous pathways that allow synthesis of pyridoxal phosphate (PLP) in a strain of E. coli that lacks 4-phosphoerythronate dehydrogenase (PdxB) when one of the seven different genes is overexpressed. These genes were identified in a multicopy suppression experiment in which a library of E. coli genes (from the ASKA collection) was introduced into a ΔpdxB strain of E. coli that is unable to synthesize PLP. Surprisingly, none of the enzymes encoded by these genes has a promiscuous 4-phosphoerythronate (4PE) dehydrogenase activity that can substitute for the missing PdxB. Rather, overproduction of these enzymes appears to facilitate at least three serendipitous pathways that draw material from other metabolic pathways and feed into the normal PLP synthesis pathway downstream of the blocked step (Figure 1).
We have characterized one of these pathways in detail (Figure 3). The first step, dephosphorylation of 3-phosphohydroxypyruvate, is catalyzed by YeaB, a predicted NUDIX hydrolase of unknown function. Although catalysis is inefficient (kcat=5.7×10−5 s−1 and kcat/KM>0.028 M−1 s−1), the enzymatic rate is 4×107-fold faster than the rate of the uncatalyzed reaction, and is sufficient to support PLP synthesis when YeaB is overproduced. The second step in the pathway is decarboxylation of 3-hydroxypyruvate (3HP). Although we found two enzymes (1-deoxyxylulose-5-phosphate synthase and the catalytic domain of α-ketoglutarate dehydrogenase) that catalyze this reaction with low but respectable activity in vitro, their involvement in pathway 1 was ruled out by genetic methods. Surprisingly, the non-enzymatic rate of decarboxylation of 3HP appears to be sufficient to support PLP synthesis. The third step in the pathway, condensation of glycolaldehyde and glycine to form 4-hydroxy-L-threonine, is catalyzed by LtaE, a low-specificity threonine aldolase whose physiological role is not known. The final step, phosphorylation of 4-hydroxy-L-threonine, is catalyzed by homoserine kinase (ThrB), which is required for synthesis of threonine. The promiscuous phosphorylation of 4-hydroxy-L-threonine is 80-fold slower than the physiological phosphorylation of homoserine. The involvement of LtaE and ThrB in pathway 1 was confirmed by genetic experiments showing that overexpression of yeaB no longer restores growth of ΔpdxB strains lacking either ltaE or thrB.
Although pathway 1 is inefficient, it provides the ΔpdxB strain with the ability to grow under conditions in which survival is otherwise impossible. In general, serendipitous assembly of an inefficient pathway from promiscuous activities of available enzymes will be important whenever the pathway provides increased fitness. This might occur when a critical metabolite is no longer available from the environment, and survival depends on assembly of a new biosynthetic pathway. A second circumstance in which an inefficient serendipitous pathway might improve fitness is the appearance of a novel compound in the environment that can be exploited as a source of carbon, nitrogen or phosphorous. Finally, chemotherapeutic agents that block metabolic pathways in bacteria or cancer cells could provide selective pressure for assembly of serendipitous pathways that allow synthesis of the end product of the blocked pathway and thus a previously unappreciated source of drug resistance. In all of these cases, even an inefficient pathway can provide a selective advantage over other cells in a particular environmental niche, allowing survival and subsequent mutations that elevate the efficiency of the pathway.
Our work is consistent with the hypothesis that the recognized metabolic network of E. coli is underlain by a denser network of reactions due to promiscuous enzymes that use and generate recognized metabolites, but also unusual metabolites that normally have no physiological role. The findings reported here highlight the abundance of cryptic capabilities in the E. coli proteome that can be drawn on to generate novel pathways. Such pathways could provide a starting place for assembly of more efficient pathways, both in nature and in the hands of metabolic engineers.
Bacterial genomes encode hundreds to thousands of enzymes, most of which are specialized for particular functions. However, most enzymes have inefficient promiscuous activities, as well, that generally serve no purpose. Promiscuous reactions can be patched together to form multistep metabolic pathways. Mutations that increase expression or activity of enzymes in such serendipitous pathways can elevate flux through the pathway to a physiologically significant level. In this study, we describe the discovery of three serendipitous pathways that allow synthesis of pyridoxal-5′-phosphate (PLP) in a strain of E. coli that lacks 4-phosphoerythronate (4PE) dehydrogenase (PdxB) when one of seven different genes is overexpressed. We have characterized one of these pathways in detail. This pathway diverts material from serine biosynthesis and generates an intermediate in the normal PLP synthesis pathway downstream of the block caused by lack of PdxB. Steps in the pathway are catalyzed by a protein of unknown function, a broad-specificity enzyme whose physiological role is unknown, and a promiscuous activity of an enzyme that normally serves another function. One step in the pathway may be non-enzymatic.