Identification of the substrate specificity-conferring amino acid residues of 4-coumarate:coenzyme A ligase allows the rational design of mutant enzymes with new catalytic properties.

4-Coumarate:coenzyme A ligases (4CLs) generally use, in addition to coumarate, caffeate and ferulate as their main substrates. However, the recently cloned Arabidopsis thaliana isoform At4CL2 is exceptional because it has no appreciable activity with ferulate. On the basis of information obtained from the crystal structure of the phenylalanine-activating domain of gramicidin S-synthetase, 10 amino acid residues were identified that may form the substrate binding pocket of 4CL. Among these amino acids, representing the putative "substrate specificity motif," only one residue, Met(293), was not conserved in At4CL2, compared with At4CL1 and At4CL3, two isoforms using ferulate. Substitution of Met(293) or Lys(320), another residue of the putative substrate specificity motif, which in the predicted three-dimensional structure is located in close proximity to Met(293), by smaller amino acids converted At4CL2 to an enzyme capable of using ferulate. The activity with caffeate was not or only moderately affected. Conversely, substitution of Met(293) by bulky aromatic amino acids increased the apparent affinity (K(m)) for caffeate up to 10-fold, whereas single substitutions of Val(294) did not affect substrate use. The results support our structural assumptions and suggest that the amino acid residues 293 and 320 of At4CL2 directly interact with the 3-methoxy group of the phenolic substrate and therefore allow a first insight into the structural principles determining substrate specificity of 4CL.

Mutational analysis of 4-coumarate:CoA ligase identifies functionally important amino acids and verifies its close relationship to other adenylate-forming enzymes.

4-Coumarate:coenzyme A ligase (4CL) is a key enzyme of general phenylpropanoid metabolism which provides the precursors for a large variety of important plant secondary products, such as lignin, flavonoids, or phytoalexins. To identify amino acids important for 4CL activity, eight mutations were introduced into Arabidopsis thaliana At4CL2. Determination of specific activities and K(m) values for ATP and caffeate of the heterologously expressed and purified proteins identified four distinct classes of mutants: enzymes with little or no catalytic activity; enzymes with greatly reduced activity but wild-type K(m) values; enzymes with drastically altered K(m) values; and enzymes with almost wild-type properties. The latter class includes replacement of a cysteine residue which is strictly conserved in 4CLs and had previously been assumed to be directly involved in catalysis. These results substantiate the close relationship between 4CL and other adenylate-forming enzymes such as luciferases, peptide synthetases, and fatty acyl-CoA synthetases.

A novel phosphopantetheine:protein transferase activating yeast mitochondrial acyl carrier protein.

In Saccharomyces cerevisiae, the low molecular weight acyl carrier protein (ACP) of mitochondrial type II fatty acid synthase (FAS) and the cytoplasmic type I FAS multienzyme contain 4'-phosphopantetheine as a prosthetic group. Sequence alignment studies with the recently isolated phosphopantetheine:protein transferase (PPTase), Ppt1p, from Brevibacterium ammoniagenes revealed the yeast open reading frame, YPL148C, as a potential PPTase gene (25% identical and 43% conserved amino acids). In accordance with this similarity, pantetheinylation of mitochondrial ACP was lost upon disruption of YPL148C. In contrast, biosynthesis of cytoplasmic holo-FAS remained unaffected by this mutation. According to these characteristics, the newly identified gene was designated as PPT2. Similar to ACP null mutants, cellular lipoic acid synthesis and, hence, respiration were abolished in PPT2 deletants. ACP pantetheinylation, lipoic acid synthesis, and respiratory competence were restored upon transformation of PPT2 mutants with cloned PPT2 DNA. In vitro, holo-ACP synthesis was achieved by incubating apo-ACP with coenzyme A in the presence of purified Ppt2p. The homologous yeast enzyme could be replaced, in this assay, by the ACP synthase (EC 2.7.8.7) of Escherichia coli but not by the type I FAS-specific PPTase of B. ammoniagenes, Ppt1p. These results conform with the inability of Ppt2p to activate the cytoplasmic type I FAS complex of yeast.

Identification, isolation and biochemical characterization of a phosphopantetheine:protein transferase that activates the two type-I fatty acid synthases of Brevibacterium ammoniagenes.

Upon heterologous expression of the Brevibacterium ammoniagenes type-I fatty acid synthase FAS-A in Escherichia coli, only the pantetheine-free apoenzyme is synthesized. Activation of FAS-A to its holoform was achieved by transformation with a second B. ammoniagenes gene, PPT1, encoding a type-I FAS-specific phosphopantetheine transferase. PPT1 was identified as a coding sequence located immediately downstream of the second FAS gene present on the B. ammoniagenes genome, fasB. Due to this linkage, PPT1 was part of the cloned fasB DNA region and, consequently, FAS-B but not FAS-A was synthesized as holoFAS in E. coli. PPT1 encodes a protein of 153 amino acids and has a calculated molecular mass of 16,884 Da. The PPT1 gene product contains 25% identical and 42% conserved amino acids compared with the type-II acyl-carrier-protein-activating enzyme of E. coli. Although there is essentially no intergenic region between fasB and PPT1, the PPTase gene is autonomously expressed in E. coli if flanked by 200 bp of its endogenous 5' DNA. The structural independence of Ppt1p was confirmed immunologically, as specific antibodies react with the purified PPTase but not with FAS-B. Overexpression and purification of the His-tagged Ppt1p allowed the in vitro activation of apoFAS-A. This holoenzyme synthesis requires, in addition to Ppt1p, CoA and Mg2+ and leads to a specific FAS activity comparable to that of natural B. ammoniagenes FAS-A. The reactivity of the in vitro-activated FAS-A was verified by the optical FAS assay and by analysis of its in vitro products. In agreement with the known overall colinearity of B. ammoniagenes FAS-B and the Saccharomyces cerevisiae FAS1 and FAS2 gene products, a PPT1-like sequence is also observed at the C terminus of FAS2. However, in contrast to B. ammoniagenes PPT1, this sequence is an integral part of the yeast FAS2 gene. Thus, activation of type-I fatty acid synthases may be accomplished by distinct trans-acting PPTase enzymes and by intrinsic cis-acting PPTase domains.

Heterologous expression and biochemical characterization of two functionally different type I fatty acid synthases from Brevibacterium ammoniagenes.

The coryneform bacterium, Brevibacterium ammoniagenes, contains two structurally related but functionally differentiated type I fatty acid synthases, FAS-A and FAS-B. Isolation of homogeneous preparations of both enzymes was achieved by constructing specific fasA and fasB expression systems. In B. ammoniagenes, insertional mutagenesis of fasB allowed the specific production of enzymatically active FAS-A. The corresponding fasA mutant was not suited for FAS-B purification as the level of this enzyme was extremely low, in the fasA-disruptants. Instead, FAS-B could be efficiently expressed in the heterologous host, Escherichia coli. Using specific antisera against each of the two FAS variants, FAS-A was shown to be the predominant FAS protein in B. ammoniagenes. In contrast the two enzymes are expressed at comparable rates in E. coli even though the same upstream sequences were associated with fasA and fasB, as in B. ammoniagenes. Due to their differential capacities of being activated to the phosphopantetheine-containing holo-enzyme in the heterologous host, only FAS-B but not FAS-A exhibited overall FAS activity when isolated from E. coli. Irrespective of their origin, the purified FAS-A and FAS-B proteins were indistinguishable with respect to their flavin fluorescence, their subunit size and their sucrose density gradient sedimentation characteristics. Nevertheless, the in vitro products of both enzymes differ characteristically: while FAS-A synthesizes mainly the 18-carbon fatty acids oleate and stearate with only traces of palmitate, the major product of FAS-B is palmitic acid. No unsaturated fatty acids are produced by FAS-B. Thus, the two B ammoniogenes type I fatty acid synthases differ, in spite of their very similar overall protein structure, in both their ability to synthesize oleic acid and in their chain-length specificities.

Identification and functional differentiation of two type I fatty acid synthases in Brevibacterium ammoniagenes.

The fatty acid synthase (FAS) from Brevibacterium ammoniagenes is a homohexameric multienzyme complex that catalyzes the synthesis of both saturated and unsaturated fatty acids. By immunological screening of a B. ammoniagenes expression library, an fas DNA fragment was isolated and subsequently used to clone the entire gene together with its flanking sequences. Within 10,525 bp of sequenced DNA, the 9,189-bp FAS coding region was identified, corresponding to a protein of 3,063 amino acids with a molecular mass of 324,910 Da. This gene (fasA) encodes, at its 5' end, the same amino acid sequence as is observed with purified B. ammoniagenes FAS. A second reading frame encoding another B. ammoniagenes FAS variant (FasB) had been identified previously. Both sequences are colinear and exhibit 61 and 47% identity at the DNA and protein levels, respectively. By using specific antibodies raised against a unique peptide sequence of FasB, this enzyme was shown to represent only 5 to 10% of the cellular FAS protein. Insertional inactivation of the FasB coding sequence causes no defective phenotype, while fasA disruptants require oleic acid for growth. Correspondingly, oleate-dependent B. ammoniagenes cells obtained by ethyl methanesulfonate mutagenesis were complemented by transformation with fasA DNA but not with fasB DNA. The data indicate that B. ammoniagenes contains two related though differently expressed type I FASs. FasA represents the bulk of cellular FAS protein and catalyzes the synthesis of both saturated and unsaturated fatty acids, while the minor variant, FasB, cannot catalyze the synthesis of oleic acid.