Holly Jessen

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

The fermentation of β‐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 α‐alanine to β‐alanine, the enzyme β‐alanyl‐CoA:ammonia lyase is induced 300‐fold (≈ 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 β‐alanine. The deduced β‐alanyl‐CoA:ammonia lyase proteins are related to putative β‐aminobutyryl‐CoA ammonia lyases involved in lysine fermentation and found in the genomes of several anaerobic bacteria. β‐Alanyl‐CoA:ammonia lyase 2 was purified to homogeneity and characterized as a heteropentamer composed of 16 kDa subunits. The apparent Km value for acryloyl‐CoA was measured as 23 ± 4 µm, independent of the concentration of the second substrate ammonia; kcat/Km was calculated as 107 m−1·s−1. The apparent Km for ammonia was much higher, 70 ± 5 mm at 150 µm acryloyl‐CoA with a much lower kcat/Km of 4 × 103 m−1·s−1. In the reverse reaction, a Km of 210 ± 30 µM was obtained for β‐alanyl‐CoA. The elimination of ammonia was inhibited by 70% at 100 mm ammonium chloride. The content of β‐alanyl‐CoA:ammonia lyase in β‐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/β‐alanine or d‐lactate/3‐hydroxypropionate.

Enzymatic Activation of Lysine 2,3-Aminomutase from Porphyromonas gingivalis

ABSTRACT The development of lysine 2,3-aminomutase as a robust biocatalyst hinges on the development of an in vivo activation system to trigger catalysis. This is the first report to show that, in the absence of chemical reductants, lysine 2,3-aminomutase activity is dependent upon the presence of flavodoxin, ferredoxin, or flavodoxin-NADP+ reductase.