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.
Herrmann
Gloria
G
Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität, Marburg, Germany.
Selmer
Thorsten
T
Jessen
Holly J
HJ
Gokarn
Ravi R
RR
Selifonova
Olga
O
Gort
Steve J
SJ
Buckel
Wolfgang
W
D000642:Ammonia-Lyases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D007700:Kinetics; D010449:Peptide Mapping; D013379:Substrate Specificity
D017931:DNA Primers; D000642:Ammonia-Lyases