•Screening efforts have revealed enzymes with over 100 promiscuous activities.•High-throughput screening for promiscuous activities can be accomplished in droplets containing single cells.•Activity-based metabolic profiling can be used to detect promiscuous activities.•Promiscuous activities may be inefficient because substrates bind in a poor position.•Inefficient activity can also result from binding to an infrequent enzyme conformation.Most, if not all, enzymes are capable of catalyzing physiologically irrelevant secondary reactions — termed ‘promiscuous’ reactions — in addition to the reactions that they have evolved to catalyze. Promiscuous activities can provide the starting point for evolution of new enzymes, both in nature and in the hands of protein engineers. Recent work suggests that the universe of promiscuous activities available in nature is enormous. New high-throughput approaches have significantly advanced our ability to identify promiscuous activities, setting the stage for synthetic biology efforts to construct novel pathways using catalysts derived from promiscuous enzymes via directed evolution.Download high-res image (131KB)Download full-size image

Pentachlorophenol (PCP) hydroxylase, the first enzyme in the pathway for degradation of PCP in Sphingobium chlorophenolicum, is an unusually slow flavin-dependent monooxygenase (kcat = 0.02 s–1) that converts PCP to a highly reactive product, tetrachlorobenzoquinone (TCBQ). Using stopped-flow spectroscopy, we have shown that the steps up to and including formation of TCBQ are rapid (5–30 s–1). Before products can be released from the active site, the strongly oxidizing TCBQ abstracts an electron from a donor at the active site, possibly a cysteine residue, resulting in an off-pathway diradical state that only slowly reverts to an intermediate capable of completing the catalytic cycle. TCBQ reductase, the second enzyme in the PCP degradation pathway, rescues this nonproductive complex via two fast sequential one-electron transfers. These studies demonstrate how adoption of an ancestral catalytic strategy for conversion of a substrate with different steric and electronic properties can lead to subtle yet (literally) radical changes in enzymatic reaction mechanisms.

Facile DNA sequencing became possible decades after many enzymes had been purified and characterized. Consequently, there are still “orphan” enyzmes for which activities are known but for which encoding genes have not been identified. Identification of the genes encoding orphan enzymes is important because it allows correct annotation of genes of unknown function or with misassigned function. Bis-γ-glutamylcystine reductase (GCR) is an orphan protein that was purified in 1988. This enzyme catalyzes the reduction of bis-γ-glutamylcystine. γ-Glutamylcysteine is the major low-molecular weight thiol in halobacteria. We purified GCR from Halobacterium sp. NRC-1 and identified the sequence of 23 tryptic peptides by nano-liquid chromatography electrospray ionization tandem mass spectrometry. These peptides cover 62% of the protein predicted to be encoded by a gene in Halobacterium sp. NRC-1 that is annotated as mercuric reductase. GCR and mercuric reductase activities were assayed using enzyme that was expressed in Escherichia coli and refolded from inclusion bodies. The enzyme had robust GCR activity but no mercuric reductase activity. The genomes of most, but not all, halobacteria for which whole genome sequences are available have close homologues of GCR, suggesting that there is more to be learned about the low-molecular weight thiols used in halobacteria.

Pyruvate is an important “hub” metabolite that is a precursor for amino acids, sugars, cofactors, and lipids in extant metabolic networks. Pyruvate has been produced under simulated hydrothermal vent conditions from alkyl thiols and carbon monoxide in the presence of transition metal sulfides at 250 °C [Cody GD et al. (2000) Science 289(5483):1337–1340], so it is plausible that pyruvate was formed in hydrothermal systems on the early earth. We report here that pyruvate reacts readily in the presence of transition metal sulfide minerals under simulated hydrothermal vent fluids at more moderate temperatures (25–110 °C) that are more conducive to survival of biogenic molecules. We found that pyruvate partitions among five reaction pathways at rates that depend upon the nature of the mineral present; the concentrations of H2S, H2, and NH4Cl; and the temperature. In most cases, high yields of one or two primary products are found due to preferential acceleration of certain pathways. Reactions observed include reduction of ketones to alcohols and aldol condensation, both reactions that are common in extant metabolic networks. We also observed reductive amination to form alanine and reduction to form propionic acid. Amino acids and fatty acids formed by analogous processes may have been important components of a protometabolic network that allowed the emergence of life.

Microbes in contaminated environments often evolve new metabolic pathways for detoxification or degradation of pollutants. In some cases, intermediates in newly evolved pathways are more toxic than the initial compound. The initial step in the degradation of pentachlorophenol by Sphingobium chlorophenolicum generates a particularly reactive intermediate; tetrachlorobenzoquinone (TCBQ) is a potent alkylating agent that reacts with cellular thiols at a diffusion-controlled rate. TCBQ reductase (PcpD), an FMN- and NADH-dependent reductase, catalyzes the reduction of TCBQ to tetrachlorohydroquinone. In the presence of PcpD, TCBQ formed by pentachlorophenol hydroxylase (PcpB) is sequestered until it is reduced to the less toxic tetrachlorohydroquinone, protecting the bacterium from the toxic effects of TCBQ and maintaining flux through the pathway. The toxicity of TCBQ may have exerted selective pressure to maintain slow turnover of PcpB (0.02 s−1) so that a transient interaction between PcpB and PcpD can occur before TCBQ is released from the active site of PcpB.

Several strains of Sphingobium chlorophenolicum have been isolated from soil that was heavily contaminated with pentachlorophenol (PCP), a toxic pesticide introduced in the 1930s. S. chlorophenolicum appears to have assembled a poorly functioning pathway for degradation of PCP by patching enzymes recruited via two independent horizontal gene transfer events into an existing metabolic pathway. Flux through the pathway is limited by PCP hydroxylase. PCP hydroxylase is a dimeric protein that belongs to the family of flavin-dependent phenol hydroxylases. In the presence of NADPH, PCP hydroxylase converts PCP to tetrachlorobenzoquinone (TCBQ). The kcat for PCP (0.024 s–1) is very low, suggesting that the enzyme is not well evolved for turnover of this substrate. Structure–activity studies reveal that substrate binding and activity are enhanced by a low pKa for the phenolic proton, increased hydrophobicity, and the presence of a substituent ortho to the hydroxyl group of the phenol. PCP hydroxylase exhibits substantial uncoupling; the C4a-hydroxyflavin intermediate, instead of hydroxylating the substrate, can decompose to produce H2O2 in a futile cycle that consumes NADPH. The extent of uncoupling varies from 0 to 100% with different substrates. The extent of uncoupling is increased by the presence of bulky substituents at position 3, 4, or 5 and decreased by the presence of a chlorine in the ortho position. The effectiveness of PCP hydroxylase is additionally hindered by its promiscuous activity with tetrachlorohydroquinone (TCHQ), a downstream metabolite in the degradation pathway. The conversion of TCHQ to TCBQ reverses flux through the pathway. Substantial uncoupling also occurs during the reaction with TCHQ.

Evolution or engineering of novel metabolic pathways can endow microbes with new abilities to degrade anthropogenic pollutants or synthesize valuable chemicals. Most studies of the evolution of new pathways have focused on the origins and quality of function of the enzymes involved. However, there is an additional layer of complexity that has received less attention. Introduction of a novel pathway into an existing metabolic network can result in inhibitory cross-talk due to adventitious interactions between metabolites and macromolecules that have not previously encountered one another. Here, we report a thorough examination of inhibitory cross-talk between a novel metabolic pathway for synthesis of pyridoxal 5′-phosphate and the existing metabolic network of Escherichia coli. We demonstrate multiple problematic interactions, including (i) interference by metabolites in the novel pathway with metabolic processes in the existing network, (ii) interference by metabolites in the existing network with the function of the novel pathway, and (iii) diversion of metabolites from the novel pathway by promiscuous activities of enzymes in the existing metabolic network. Identification of the mechanisms of inhibitory cross-talk can reveal the types of adaptations that must occur to enhance the performance of a novel metabolic pathway as well as the fitness of the microbial host. These findings have important implications for evolutionary studies of the emergence of novel pathways in nature as well as genetic engineering of microbes for “green” manufacturing processes.

Photolabile 3′,5′-dimethoxybenzoin-based linkers are advantageous for a variety of solid-phase synthetic procedures and manipulations of biomolecules because UV irradiation in aqueous media provides fast and essentially quantitative release of tethered molecules, while generating unreactive side products. Practical applications of previously reported linkers are compromised to some extent by the 1,3-dithiane protection of the benzoin carbonyl group and the lengthy synthesis. We have extended the group of photocleavable 3′,5′-dimethoxybenzoin-based linkers by designing and synthesizing a linker in which the carbonyl group is protected as a dimethyl ketal. This protection is compatible with commonly used esterification and amide bond formation techniques, including the Fmoc/tBu strategy for solid phase peptide synthesis, is stable under mild acidic conditions, and can be quantitatively removed in <5 min by 3% TFA in dichloromethane. Irradiation of beads carrying peptides attached to the linker at 350 nm in aqueous or partially aqueous media affords >90% release after 30 min. The linker was synthesized from commercially available starting materials in five steps with an overall yield of 40% and without any column chromatography purification. Additionally, we developed a route to a dithiane-protected linker that requires only two steps and proceeds in 65% yield, a significant improvement over previous synthetic routes.