P lab

Where Do New Enzymes Come From?

We are interested in elucidating the biochemistry that underlies enzyme evolution. New biochemical functions can emerge as organisms adapt to changing environments. For example, microorganisms evolved the ability to degrade man-made pesticides (such as paraoxon) in less than 50 years; not forgetting that antibiotic resistance evolved within 3 years of penicillin being introduced into the clinic.

Promiscuity as the kick-start. The evolutionary origins of new activities, as well as the pathways for their subsequent optimization, remain poorly understood. We (and others) have demonstrated that enzymes showing catalytic promiscuity and/or substrate ambiguity can provide starting points for re-evolving old activities (i.e. rescuing auxotrophic strains). Recently, we have taken the next step: we have tested the ability of promiscuous enzymes to impart genuinely new phenotypes, such as growth on new nutrients, toxin resistance, etc.

The library-on-library screen. A library of Escherichia coli clones, each over-expressing a single ORF, was pooled and exposed to a collection of 2,000 novel growth environments in microplates (figure, right). We screened for the cells that could survive by metabolizing the novel nutrient or toxin found in each environment. Our results showed that phenotypic innovation mediated by protein over-expression can be very common. Further, we have identified promiscuous proteins – from a common, lab-friendly bacterium – that enabled increased growth or resistance in approximately 500 of the growth scenarios. We are characterizing selected examples in detail. For example, over-expression of the previously-uncharacterized protein YcgZ increases resistance to a cephalosoprin antibiotic, cefuroxime (insets on right).

This work offers insight into the fundamental processes that underlie the evolution of new metabolic pathways. It will be possible to apply the same technologies to pathogenic microorganisms, in order to understand the infection process. We are excited by the possibility of using these results to shed light on applied problems such as bioremediation and bio-manufacturing (see Synthetic Biology) and potentially, enzyme engineering technologies (see DNA Ligases).

Biolog plate

The evolution of PLP-dependent enzymes. Pyridoxal 5'-phosphate (PLP) is a versatile cofactor, used by many enzymes involved in amino acid metabolism. PLP-dependent enzymes are excellent candidates to study the evolution of old and new activities, as they are structurally and catalytically diverse. However, the reactivity of the cofactor also gives rise to catalytic promiscuity and/or recognition of alternate substrates. It is not uncommon for several non-homologous PLP-dependent enzymes to catalyze the same reaction (albeit often only weakly). We are utilizing site-directed and random mutagenesis approaches to explore mutations that control catalytic specificities, and how activities trade off under selective conditions. In the long run, we hope to gain new insights into the evolution of the various PLP-binding scaffolds.

The PLP-dependent enzyme cystathionine β-lyase possesses promiscuous alanine racemase activity. Error-prone PCR and genetic selection have been used to identify mutations near the active site that increase the promiscuous activity.