Biochemical and molecular characterization of phenylacetate-coenzyme A ligase, an enzyme catalyzing the first step in aerobic metabolism of phenylacetic acid in Azoarcus evansii

Abstract

Phenylacetate-coenzyme A ligase (PA-CoA ligase; AMP forming, EC 6.2. 1.30), the enzyme catalyzing the first step in the aerobic degradation of phenylacetate (PA) in Azoarcus evansii, has been purified and characterized. The gene (paaK) coding for this enzyme was cloned and sequenced. The enzyme catalyzes the reaction of PA with CoA and MgATP to yield phenylacetyl-CoA (PACoA) plus AMP plus PPi. The enzyme was specifically induced after aerobic growth in a chemically defined medium containing PA or phenylalanine (Phe) as the sole carbon source. Growth with 4-hydroxyphenylacetate, benzoate, adipate, or acetate did not induce the synthesis of this enzyme. This enzymatic activity was detected very early in the exponential phase of growth, and a maximal specific activity of 76 nmol min(-1) mg of cell protein(-1) was measured. After 117-fold purification to homogeneity, a specific activity of 48 micromol min(-1) mg of protein(-1) was achieved with a turnover number (catalytic constant) of 40 s(-1). The protein is a monomer of 52 kDa and shows high specificity towards PA; other aromatic or aliphatic acids were not used as substrates. The apparent K(m) values for PA, ATP, and CoA were 14, 60, and 45 microM, respectively. The PA-CoA ligase has an optimum pH of 8 to 8.5 and a pI of 6.3. The enzyme is labile and requires the presence of glycerol for stabilization. The N-terminal amino acid sequence of the purified protein showed no homology with other reported PA-CoA ligases. The gene encoding this enzyme is 1, 320 bp long and codes for a protein of 48.75 kDa (440 amino acids) which shows high similarity with other reported PA-CoA ligases. An amino acid consensus for an AMP binding motif (VX2SSGTTGXP) was identified. The biochemical and molecular characteristics of this enzyme are quite different from those of the isoenzyme catalyzing the same reaction under anaerobic conditions in the same bacterium.

FIG. 1

Aerobic and anaerobic catabolic pathways of phenylacetic acid in A. evansii and T. aromatica. I, phenylacetic acid; II, PACoA; III, phenylglyoxyl-CoA; IV, phenylglyoxylate; V, benzoyl-CoA; CoASH, coenzyme A.

FIG. 2

In vivo formation of [¹⁴C]PACoA in A. evansii cells grown aerobically in the presence of [¹⁴C]PA (A) or [¹⁴C]phenylalanine (B). The reactions were stopped with 10% formic acid and separated by TLC followed by autoradiography. Lanes 1 to 4, samples taken at 0 and 30 s and 1 and 2 min in the presence of PA; lanes 5 to 8, samples taken at 30 s and 1, 2, and 5 min in the presence of phenylalanine. X indicates an unknown product.

FIG. 3

SDS-PAGE of PA-CoA ligase protein fractions collected during the purification scheme. Lanes 1 and 7, molecular mass protein standards containing phosphorylase (97 kDa), bovine serum albumin (67 kDa), ovalbumin (45 kDa), lactate dehydrogenase (34 kDa), and carbonic anhydrase (29 kDa); lane 2, crude extract (supernatant after centrifugation at 100,000 × g, 130 μg of protein); lane 3, DEAE fraction (negative chromatography, 85 μg of protein); lane 4, DEAE fraction (positive chromatography, 60 μg of protein); lane 5, Q-Sepharose fraction (45 μg of protein); lane 6, reactive-green fraction (6 μg of protein); lane 8, hydroxyapatite fraction (5 μg of protein).

FIG. 4

(A) HPLC separation of PA-CoA ligase reaction product. (B) Results of TLC and autoradiography of PA-CoA ligase in the presence of [¹⁴C]PA. Lane 1, acid-stopped sample after 1 min; lane 2, the same sample after alkaline hydrolysis.

FIG. 5

Amino acid alignment of PA-CoA ligases from different microorganisms. Ae, A. evansii; Ec, E. coli (9); Pp, P. putida (33); Psp, Pseudomonas sp. strain Y2 (44); Bh, B. halodurans (42); MtF390, coenzyme F₃₉₀ M. thermoautotrophicum (40). The Arabic numbers refer to the amino acid sequence deduced from the nucleotide sequence. The Roman numerals above the sequence denote postulated binding motifs. I, AMP-binding motif; II and III, postulated substrate-binding site motifs. Note that only the A. evansii and P. putida genes have been proven to code for this enzyme.