Phenylacetate metabolism in thermophiles: characterization of phenylacetate-CoA ligase, the initial enzyme of the hybrid pathway in Thermus thermophilus.
Phenylacetate-CoA ligase (E.C. 6.2.1.30), the initial enzyme in the metabolism of phenylacetate, was studied in Thermus thermophilus strain HB27. Enzymatic activity was upregulated during growth on phenylacetate or phenylalanine. The phenylacetate-CoA ligase gene (paaK) was cloned and heterologously expressed in Escherichia coli and the recombinant protein was purified. The enzyme catalyzed phenylacetate + CoA + MgATP --> phenylacetyl-CoA + AMP + MgPP(i) with a V(max) of 24 micromol/min/mg protein at a temperature optimum of 75 degrees C. The apparent K(m) values for ATP, CoA, and phenylacetate were 6, 30, and 50 microM: , respectively. The protein was highly specific toward phenylacetate and showed only low activity with 4-hydroxyphenylacetate. Despite an amino acid sequence identity of >50% with its mesophilic homologues, phenylacetate-CoA ligase was heat stable. The genome contained further homologues of genes, which are postulated to be involved in the CoA ester-dependent metabolic pathway of phenylacetate (hybrid pathway). Enzymes of this thermophile are expected to be robust and might be useful for further studies of this yet unresolved pathway.
Erb
Tobias J
TJ
Institut für Biologie II-Mikrobiologie, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany.
Ismail
Wael
W
Fuchs
Georg
G
D003066:Coenzyme A Ligases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010648:Phenylacetates / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D016963:Thermus thermophilus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
D010648:Phenylacetates; D011993:Recombinant Fusion Proteins; D003066:Coenzyme A Ligases; C023914:phenylacetate - CoA ligase; C025136:phenylacetic acid