Table 1.

Strategies for enzyme function discovery

Techniques available	Enzyme/genetic requirements	Purposes	Advantages	Inconveniences	Key technologies
In vitro activity-based metabolomic profiling	Purified, homogeneous enzyme	Track enzyme-induced changes in a complex metabolite extract	High throughput (hundreds to thousands of metabolites can be screened).	Enzymes have to be purified to homogeneity.	Protein purification
			Physiologically relevant library of substrates and co-factors.	Host organism or related species has to be cultured.	LC/GC/CE-MS
			No a priori knowledge of the types and number of substrates and products involved.	Recombinant expression might lead to loss of native partner or post-translational modifications required for activity.	NMR
			No a priori knowledge of the type of chemistry catalysed.	Substrates might not be present at quantifiable levels in molecular extract.	Libraries of spectral data
			Direct identification of potential substrates and products.
Ex vivo metabolomic profiling – genetically modified/chemically treated organism	None or verified genetic knockout/over-expression strain of organism of interest	Identify one enzymatic reaction or pathway that is disturbed upon deletion/alteration of levels of a particular enzyme	High throughput (hundreds to thousands of metabolites can be screened).	Host organism or related species has to be cultured and genetically tractable.	Genetic manipulation LC/GC/CE-MS
			No knowledge of the types and number of substrates and products involved required.	Candidate substrates and products might constitute secondary effect changes.	NMR
			No knowledge of the type of chemistry catalysed required.	Levels of substrates/products might be tightly controlled and not change.	Libraries of spectral data
			No enzyme purification required	Chemical with a clear phenotype must be available.
			Preservation of native enzyme partners and post-translational modifications.
Activity-based protein profiling	None	Track activity of a specific class of enzymes towards a probe	High throughput (several dozen enzymes can be screened).	Highly selective and specific probe needs to be synthesized.	Chemical probe
			Identifies active enzymes.	Identification of physiological substrates needs to be carried out subsequently.	Gel electrophoresis
			Highly specific for the chemistry and enzyme class to which the probe has been developed.	Host organism or related species has to be cultured.	Imaging
			No enzyme purification or genetic modification required	Active enzyme of interest needs to be identified.	Protein identification
			Preservation of native enzyme partners and post-translational modifications.
Computational enzymology	High-resolution structure	Identification of putative substrates, products and intermediates based on structural determinants	High throughput in silico approach can be applied to any enzyme type.	Relies on strength of ligand docking software and accuracy of crystal structure.	Docking
			No a priori knowledge of substrate specificity or type of chemistry catalysed required.	Identified compounds might not exist in the host organism.	Virtual libraries
					Computation
X-ray crystallography	Purified, homogeneous enzyme	Identify co-purified small molecules associated with purified enzyme	Tightly bound ligands can directly lead to the identity of substrates/products/intermediates.	Enzymes have to co-purify with a tightly bound metabolite.	Protein purification
	High-resolution structure			Enzymes have to be crystallized and the structure has to be solved at sufficiently high resolution.	Crystallization
				Bound ligand structure has to be determined.	Structure determination
				Bound ligand might not be present in the host organism or be related to the native function.

NMR, nuclear magnetic resonance; GC/LC/CE, gas or liquid chromatography or capillary electrophoresis.