Discovery of enzymatic activity using stable isotope metabolite labeling and liquid chromatography-mass spectrometry.

Stable isotope labeling of an intracellular chemical precursor or metabolite allows direct detection of downstream metabolites of that precursor, arising from novel enzymatic activity of interest, using metabolite profiling liquid chromatography-mass spectrometry. This approach allows the discrimination of downstream metabolites produced from novel enzymatic activity from the unlabeled form of the metabolite arising from native metabolic processes within the cell. Even for the case in which a given product of novel enzymatic activity is a transient, the novel enzymatic activity that produced it can be demonstrated to exist indirectly by identification of product-specific downstream metabolites. Therefore, direct or indirect discovery of novel enzymatic machinery engineered within a cell can be accomplished without a requirement for direct product purification or identification. The application of this approach to confirm the presence of a novel metabolic activity, alanine 2,3-aminomutase, obtained by mutagenesis and selection are discussed. The advantages of metabolite profiling approaches to metabolic engineering in terms of accelerating enzyme discovery and development of intellectual property will also be highlighted.

Two beta-alanyl-CoA:ammonia lyases in Clostridium propionicum.

The fermentation of beta-alanine by Clostridium propionicum proceeds via activation to the CoA-thiol ester, followed by deamination to acryloyl-CoA, which is also an intermediate in the fermentation of l-alanine. By shifting the organism from the carbon and energy source alpha-alanine to beta-alanine, the enzyme beta-alanyl-CoA:ammonia lyase is induced 300-fold (approximately 30% of the soluble protein). The low basal lyase activity is encoded by the acl1 gene, whereas the almost identical acl2 gene (six amino acid substitutions) is responsible for the high activity after growth on beta-alanine. The deduced beta-alanyl-CoA:ammonia lyase proteins are related to putative beta-aminobutyryl-CoA ammonia lyases involved in lysine fermentation and found in the genomes of several anaerobic bacteria. beta-Alanyl-CoA:ammonia lyase 2 was purified to homogeneity and characterized as a heteropentamer composed of 16 kDa subunits. The apparent K(m) value for acryloyl-CoA was measured as 23 +/- 4 microm, independent of the concentration of the second substrate ammonia; k(cat)/K(m) was calculated as 10(7) m(-1) x s(-1). The apparent K(m) for ammonia was much higher, 70 +/- 5 mm at 150 microm acryloyl-CoA with a much lower k(cat)/K(m) of 4 x 10(3) m(-1) x s(-1). In the reverse reaction, a K(m) of 210 +/- 30 microM was obtained for beta-alanyl-CoA. The elimination of ammonia was inhibited by 70% at 100 mm ammonium chloride. The content of beta-alanyl-CoA:ammonia lyase in beta-alanine grown cells is about 100 times higher than that required to sustain the growth rate of the organism. It is therefore suggested that the enzyme is needed to bind acryloyl-CoA, in order to keep the toxic free form at a very low level. A formula was derived for the calculation of isomerization equilibra between L-alanine/beta-alanine or D-lactate/3-hydroxypropionate.

Separation and identification of organic acid-coenzyme A thioesters using liquid chromatography/electrospray ionization-mass spectrometry.

A method has been developed for the direct determination of coenzyme A (CoA) and organic acid-CoA thioesters in mixtures using directly combined liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS). Mixtures of CoA and organic acid-CoA thioesters were analyzed by LC/ESI-MS with detection of protonated molecular ions and characteristic fragment ions for each compound. The identities of the CoA-thioesters were established based on LC retention times and simultaneously recorded mass spectra. Monitoring of the CoA specific fragment ion at m/z 428 throughout the chromatogram provides a unique fingerprint for CoA content in the samples that corroborates the identification of organic acid-CoA thioesters in the mixtures. Furthermore, fragment ions arising from the ester linkage portion of the molecule allow unambiguous identification of the CoA esters in the samples. A second LC elution system was developed that allows the simultaneous separation and identification of 2-hydroxypropionyl-CoA (lactyl-CoA) and 3-hydroxypropionyl CoA (3HP-CoA), which have the same mass and identical MS fragmentation behavior. The utility of LC/ESI-MS employing this elution system is demonstrated by the determination of 3HP-CoA and lactyl-CoA (converted to CoA-thioesters from their corresponding free acids using CoA-transferase) in fermentation broths from Escherichia coli strains engineered for the production of 3-hydroxypropionic acid (3HP). External calibration employing a purified 3HP-CoA standard allowed indirect quantification of 3HP content in the broth with a precision of 1% (RSD). The feasibility of extending the method described above to perform LC/selected reaction monitoring-tandem mass spectrometry for direct determination of organic acid-CoA thioesters in cells was also demonstrated.