Malonyl CoA-acyl Carrier Protein Transacylase Research Articles

Cloning and Enzymatic Activity Analysis of the Malonyl-CoA:acyl Carrier Protein Transacylase in <i>Brassica napus</i> Cultivars with Different Oil Content

The full-length genomic DNA of MCAT (Malonyl-CoA:acyl carrier protein transacylase) in Brassica napus was cloned. BnMCAT shares very high identity with AtMCAT in gene sequence and gene structure. A multiple alignment of the protein sequence showed that BnMCAT shares high identity with other MCATs from E. coli and plants. BnMCAT was expressed in all tissues, such as roots, stems, leaves, flowers, and seeds, and no significant differences in the expression level were found in different embryo stages after pollination. According to an in vitro relative activity analysis, purified recombinant BnMCAT expressed in E. coli had transacylase activity. Although the relative activities of BnMCAT in crude extracts isolated from different staged embryos were similar and showed little variation, a higher relative activity was found in a crude extract isolated from embryos in comparison to leaves. Different relative activities of BnMCAT in crude extracts isolated from cultivars with different oil content were also found, suggesting that the activity of BnMCAT might be a decisive factor for a high oil content. Together, these results showed that BnMCAT is an important enzyme in the FAS system and indicate that BnMCAT might be a new target enzyme for future crop improvement through genetic engineering.

Cloning and stress-responding expression analysis of malonyl CoA-acyl carrier protein transacylase gene of Nannochloropsis gaditana

Malonyl CoA-acyl carrier protein transacylase (MCAT, E2.3.1.39) is closely associated with the FASII pathway of fatty acid biosynthesis. However, the information about microalgal MCAT is scarce. In this study, a MCAT gene was isolated from Nannochloropsis gaditana with its deduced protein (NgMCAT) characterized with bioinformatic tools. The abundance of the NgMCAT transcript under different environmental conditions was determined with real-time quantitative PCR. Results showed that the open reading frame (ORF) of NgMCAT was 1059bp in length, which encoded 352 amino acid residues. The abundance of NgMCAT transcript reached the maximum (5.17-fold) at 6h when sodium nitrate was limited, and reached the maximum (4.25-fold) at 12h at low temperature (15°C). The abundance of NgMCAT transcript fluctuated at high temperature (35°C) when the concentration of nitrate and sodium chloride exceeded 150mg/L and 62g/L, respectively. In addition, some components of fatty acid that changed with the expression of NgMCAT were also observed.

Cloning and functional analysis of putative malonyl-CoA:acyl-carrier protein transacylase gene from the docosahexaenoic acid-producer Schizochytrium sp. TIO1101

Malonyl-CoA:acyl-carrier protein transacylase (MCAT), which transfers the malonyl group from malonyl-CoA to holo-acyl carrier protein (ACP), is a key enzyme in fatty acid biosynthesis. Schizochytrium sp. TIO1101 is a marine protist with high levels of docosahexaenoic acid accumulation. In this study, the putative fabD gene coding MCAT was isolated from Schizochytrium sp. TIO1101. The Schizochytrium MCAT gene (ScTIOfabD) contained an 1176bp open reading frame encoding a protein of 391 amino acids. The ScTIOfabD gene exhibited high novelty in nucleotide and amino acid sequence. The highest amino acid identity was only 35% between ScTIOMCAT and the reported MCATs. Further studies demonstrated that ScTIOMCAT could bind malonyl-CoA directly and transfer malonyl group from malonyl-CoA to the ACP domain in vitro. Phylogenetic analysis suggested that ScTIOMCAT was relative close to MCATs of yeast strains. Overexpression of ScTIOMCAT in Saccharomyces cereviseae significantly increased the MCAT activity, without negative effects on the growth rate of the host strain. In addition, ScTIOMCAT generated 16.8 and 62% increase in biomass and fatty acid accumulation, respectively, and did not alter the profile of fatty acid. Our results indicated that the novel MCAT gene from Schizochytrium sp. TIO1101 was crucial for fatty acid synthesis and had potential applications for genetic modifications of oil-producing species.

Compromised Mitochondrial Fatty Acid Synthesis in Transgenic Mice Results in Defective Protein Lipoylation and Energy Disequilibrium

A mouse model with compromised mitochondrial fatty acid synthesis has been engineered in order to assess the role of this pathway in mitochondrial function and overall health. Reduction in the expression of mitochondrial malonyl CoA-acyl carrier protein transacylase, a key enzyme in the pathway encoded by the nuclear Mcat gene, was achieved to varying extents in all examined tissues employing tamoxifen-inducible Cre-lox technology. Although affected mice consumed more food than control animals, they failed to gain weight, were less physically active, suffered from loss of white adipose tissue, reduced muscle strength, kyphosis, alopecia, hypothermia and shortened lifespan. The Mcat-deficient phenotype is attributed primarily to reduced synthesis, in several tissues, of the octanoyl precursors required for the posttranslational lipoylation of pyruvate and α-ketoglutarate dehydrogenase complexes, resulting in diminished capacity of the citric acid cycle and disruption of energy metabolism. The presence of an alternative lipoylation pathway that utilizes exogenous free lipoate appears restricted to liver and alone is insufficient for preservation of normal energy metabolism. Thus, de novo synthesis of precursors for the protein lipoylation pathway plays a vital role in maintenance of mitochondrial function and overall vigor.

Functional characterizations of malonyl-CoA:acyl carrier protein transacylase (MCAT) in Eimeria tenella

Eimeria tenella, an apicomplexan parasite in chickens, possesses an apicoplast and its associated metabolic pathways including the Type II fatty acid synthesis (FAS II). Malonyl-CoA:acyl-carry protein transacylase (MCAT) encoded by the fabD gene is one of the essential enzymes in the FAS II system. In the present study, the entire E. tenella MCAT gene (EtfabD) was cloned and sequenced. Immunolabeling located this protein in the apicoplast organelle in coccidial sporozoites. Functional replacement of the fabD gene with amber mutation of E. coli temperature-sensitive LA2-89 strain by E. tenella EtMCAT demonstrated that EcFabD and EtMCAT perform the same biochemical function. The recombinant EtMCAT protein was expressed and its general biochemical features were also determined. An alkaloid natural product corytuberine (CAS: 517-56-6) could specifically inhibit the EtMCAT activity (IC50=16.47μM), but the inhibition of parasite growth in vitro by corytuberine was very weak (the predicted MIC50=0.65mM).

Crystal structure of malonyl CoA-Acyl carrier protein transacylase from Xanthomanous oryzae pv. oryzae and its proposed binding with ACP.

Xanthomonas oryzae pv. oryzae (Xoo) is a plant bacterial pathogen that causes bacterial blight (BB) disease, resulting in serious production losses of rice. The crystal structure of malonyl CoA-acyl carrier protein transacylase (XoMCAT), encoded by the gene fabD (Xoo0880) from Xoo, was determined at 2.3 Å resolution in complex with N-cyclohexyl-2-aminoethansulfonic acid. Malonyl CoA-acyl carrier protein transacylase transfers malonyl group from malonyl CoA to acyl carrier protein (ACP). The transacylation step is essential in fatty acid synthesis. Based on the rationale, XoMCAT has been considered as a target for antibacterial agents against BB. Protein-protein interaction between XoMCAT and ACP was also extensively investigated using computational docking, and the proposed model revealed that ACP bound to the cleft between two XoMCAT subdomains.

Improving fatty acid production in escherichia coli through the overexpression of malonyl coA‐Acyl carrier protein transacylase

The microbial biosynthesis of free fatty acid, which can be used as precursors for the production of fuels or chemicals from renewable carbon sources, has attracted significant attention in recent years. Free fatty acids can be produced by introducing an acyl-carrier protein (ACP) thioesterase (TE) gene into Escherichia coli. The first committed step of fatty acid biosynthesis is the conversion of acetyl-CoA to malonyl-CoA by an adenosine triphosphate (ATP)-dependent acetyl-CoA carboxylase followed by the conversion of malonyl-CoA to malonyl-ACP through the enzyme malonyl CoA-acyl carrier protein transacylase (MCT; FabD). The E. coli fabD gene encoding MCT has been cloned and studied. However, the effect of FabD overexpression in a fatty acid overproducing strain has not been examined. In this study, we examined the effect of FabD overexpression in a fatty acid overproducing strain carrying an acyl-ACP TE. Specifically, the effect of overexpressing a fabD gene from four different organisms on fatty acid production was compared. The strains carrying a fabD gene from E. coli, Streptomyces avermitilis MA-4680, or Streptomyces coelicolor A3(2) improved the free fatty acid production; these three strains produced more free fatty acids, about 11% more, than the control strain. The strain carrying a fabD gene from Clostridium acetobutylicum ATCC 824, however, produced similar quantities of free fatty acids as the control strain. In addition, the three FabD overexpressed strains also have higher fatty acid/glucose yields. The results suggested that FabD overexpression can be used to improve free fatty acid production by increasing the malonyl-ACP availability.

New design platform for malonyl-CoA-acyl carrier protein transacylase

Malonyl-CoA-acyl carrier protein transacylase (MCAT) transfers the malonyl group from malonyl-CoA to holo-acyl carrier protein (ACP), and since malonyl-ACP is a key building block for fatty-acid biosynthesis it is considered as a promising antibacterial target. The crystal structures of MCAT from Staphylococcus aureus and Streptococcus pneumoniae have been determined at 1.46 and 2.1Å resolution, respectively. In the SaMCAT structure, the N-terminal expression peptide of a neighboring molecule running in the opposite direction of malonyl-CoA makes extensive interactions with the highly conserved “Gly-Gln-Gly-Ser-Gln” stretch, suggesting a new design platform. Mutagenesis results suggest that Ser91 and His199 are the catalytic dyad.

Cloning, purification, crystallization and preliminary X-ray crystallographic analysis of MCAT fromStaphylococcus aureus

Malonyl-CoA:acyl-carrier protein transacylase (MCAT), encoded by the fabd gene, is a key enzyme in type II fatty-acid biosynthesis. It is responsible for transferring the malonyl group from malonyl-CoA to the holo acyl-carrier protein (ACP). Since the type II system differs from the type I system that mammals use, it has received enormous attention as a possible antibiotic target. In particular, only a single isoform of MCAT has been reported and a continuous coupled enzyme assay has been developed. MCAT from Staphylococcus aureus was overexpressed in Escherichia coli and the protein was purified and crystallized. Diffraction data were collected to 1.2 A resolution. The crystals belonged to space group P2(1), with unit-cell parameters a = 41.608, b = 86.717, c = 43.163 A, alpha = gamma = 90, beta = 106.330 degrees . The asymmetric unit contains one SaMCAT molecule.

SMb20651 is another acyl carrier protein from Sinorhizobium meliloti

Acyl carrier proteins (ACPs) are small acidic proteins that carry growing acyl chains during fatty acid or polyketide synthesis. In rhizobia, there are four different and well-characterized ACPs: AcpP, NodF, AcpXL and RkpF. The genome sequence of Sinorhizobium meliloti 1021 reveals two additional ORFs that possibly encode additional ACPs. One of these, smb20651, is located on the plasmid pSymB as part of an operon. The genes of the operon encode a putative asparagine synthetase (AsnB), the predicted ACP (SMb20651), a putative long-chain fatty acyl-CoA ligase (SMb20650) and a putative ammonium-dependent NAD+ synthetase (NadE1). When SMb20651 was overexpressed in Escherichia coli, [3H]beta-alanine, a biosynthetic building block of 4'-phosphopantetheine, was incorporated into the protein in vivo. The purified SMb20651 was modified with 4'-phosphopantetheine in the presence of S. meliloti holo-ACP synthase (AcpS). Also, holo-SMb20651 was modified in vitro with a malonyl group by malonyl CoA-ACP transacylase. In E. coli, coexpression of SMb20651 together with other proteins such as AcpS and SMb20650 led to the formation of additional forms of SMb20651. In this bacterium, acylation of SMb20651 with C12 : 0 or C18 : 0 fatty acids was detected, demonstrating that this protein is involved in fatty acid biosynthesis or transfer. Expression of SMb20651 was detected in S. meliloti as holo-SMb20651 and acyl-SMb20651.

Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of malonyl-CoA–acyl carrier protein transacylase (FabD) fromXanthomonas oryzaepv.oryzae

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight in rice, which is one of the most devastating diseases in rice-cultivating countries. The Xoo0880 (fabD) gene coding for a malonyl-CoA-acyl carrier protein transacylase (MCAT) from Xoo was cloned and expressed in Escherichia coli. MCAT is an essential enzyme that catalyzes a key reaction of fatty-acid synthesis in bacteria and plants: the conversion of malonyl-CoA to malonyl-acyl carrier protein. The FabD enzyme was purified and crystallized in order to elucidate its three-dimensional structure and to determine its enzymatic reaction mechanism and biological importance. The crystal obtained diffracted to 1.9 A resolution and belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 41.4, b = 74.6, c = 98.5 A. According to Matthews coefficient calculations, the crystallographic structure contains only one monomeric unit in the asymmetric unit with a V(M) of 2.21 A(3) Da(-1) and a solvent content of 44.3%.

Dissecting the Component Reactions Catalyzed by the Actinorhodin Minimal Polyketide Synthase

The actinorhodin (act) minimal polyketide synthase (PKS) from Streptomyces coelicolor consists of three proteins: an acyl carrier protein (ACP) and two beta-ketoacyl ACP synthase components known as KSalpha and KSbeta. The act minimal PKS catalyzes at least 18 separate reactions which can be divided into loading, initiation, extension, and cyclization and release phases. Two quantitative kinetic assays were developed and used to measure individual rate and Michaelis constants for loading, initiation and extension steps. In the minimal PKS, the reaction between malonyl CoA and ACP to form malonyl ACP (loading) is the rate-limiting step (kcat = 0.49 min-1, KM = 207 microM). This reaction increases 5-fold in rate in the presence of KSalphaKSbeta (kcat = 2.3 min-1, KM = 215 microM). In the presence of S. coelicolor malonyl CoA:ACP transacylase (MCAT), the rate of loading increases and the kinetic parameters of malonyl-ACP as a substrate of KSalphaKSbeta can be measured (kcat = 20.6 min-1, KM = 2.4 microM). Under these conditions, it appears that decarboxylation of malonyl-ACP to form acetyl-ACP (initiation) is the rate-limiting step. When an excess of acetyl ACP is supplied, either chain extension, cyclization, or release steps become rate limiting (k approximately 60 min-1). No ACP-bound intermediates could be observed, suggesting that partially or fully extended chains do not accumulate because chain extension is rate limiting under these conditions and that cyclization and release are fast. apo-ACP acts as a mixed inhibitor of malonyl ACP binding to KSalpha/KSbeta (Kic = 50 microM, Kiu = 137 microM), but apo-ACP does not appear to inhibit MCAT.

Structure ofMycobacterium tuberculosismtFabD, a malonyl-CoA:acyl carrier protein transacylase (MCAT)

Mycobacteria display a unique and unusual cell-wall architecture, central to which is the membrane-proximal mycolyl-arabinogalactan-peptidoglycan core (mAGP). The biosynthesis of mycolic acids, which form the outermost layer of the mAGP core, involves malonyl-CoA:acyl carrier protein transacylase (MCAT). This essential enzyme catalyses the transfer of malonyl from coenzyme A to acyl carrier protein AcpM, thus feeding these two-carbon units into the chain-elongation cycle of the type II fatty-acid synthase. The crystal structure of M. tuberculosis mtFabD, the mycobacterial MCAT, has been determined to 3.0 A resolution by multi-wavelength anomalous dispersion. Phasing was facilitated by Ni2+ ions bound to the 20-residue N-terminal affinity tag, which packed between the two independent copies of mtFabD.

Malonyl‐CoA: acyl carrier protein transacylase from Helicobacter pylori: Crystal structure and its interaction with acyl carrier protein

Malonyl-CoA: acyl carrier protein transacylase (MCAT) is a critical enzyme responsible for the transfer of the malonyl moiety to holo-acyl carrier protein (ACP) forming the malonyl-ACP intermediates in the initiation step of type II fatty acid synthesis (FAS II) in bacteria. MCAT has been considered as an attractive drug target in the discovery of antibacterial agents. In this study, the crystal structure of MCAT from Helicobacter pylori (Hp) at 2.5 angstroms resolution is reported, and the interaction of HpMCAT with HpACP is extensively investigated by using computational docking, GST-pull-down, and surface plasmon resonance (SPR) technology-based assays. The crystal structure results reveal that HpMCAT has a compact folding composed of a large subdomain with a similar core as in alpha/beta hydrolases, and a similar ferredoxin-like small subdomain as in acylphosphatases. The docking result suggests two positively charged areas near the entrance of the active site of HpMCAT as the ACP-binding region. Binding assay research shows that HpMCAT demonstrates a moderately binding ability against HpACP. The solved 3D structure of HpMCAT is expected to supply useful information for the structure-based discovery of novel inhibitors against MCAT, and the quantitative study of HpMCAT interaction with HpACP is hoped to give helpful hints in the understanding of the detailed catalytic mechanisms for HpMCAT.

Structural and Functional Studies on SCO1815: A β-Ketoacyl-Acyl Carrier Protein Reductase from Streptomyces coelicolor A3(2)

Aromatic polyketides are medicinally important natural products produced by type II polyketide synthases (PKSs). Some aromatic PKSs are bimodular and include a dedicated initiation module which synthesizes a non-acetate primer unit. Understanding the mechanism of this initiation module is expected to further enhance the potential for regiospecific modification of bacterial aromatic polyketides. A typical initiation module is comprised of a ketosynthase (KS), an acyl carrier protein (ACP), a malonyl-CoA:ACP transacylase (MAT), an acyl-ACP thioesterase, a ketoreductase (KR), a dehydratase (DH), and an enoyl reductase (ER). Thus far, the identities of the ketoreductase, dehydratase, and enoyl reductase remain a mystery because they are not encoded within the PKS biosynthetic gene cluster. Here we report that SCO1815 from Streptomyces coelicolor A3(2), an uncharacterized homologue of a NADPH-dependent ketoreductase, recognizes and reduces the beta-ketoacyl-ACP intermediate from the initiation module of the R1128 PKS. SCO1815 exhibits moderate specificity for both the acyl chain and the thiol donor. The X-ray crystal structure of SCO1815 was determined to 2.0 A. The structure shows that SCO1815 adopts a Rossmann fold and suggests that a conformational change occurs upon cofactor binding. We propose that a positively charged patch formed by three conserved residues is the ACP docking site. Our findings provide new engineering opportunities for incorporating unnatural primer units into novel polyketides and shed light on the biology of yet another cryptic protein in the S. coelicolor genome.

Mapping the active site ofEscherichia colimalonyl-CoA–acyl carrier protein transacylase (FabD) by protein crystallography

Malonyl-CoA-acyl carrier protein transacylase (FabD; EC 2.3.1.39) is a key enzyme in the fatty-acid biosynthesis pathway of bacteria, catalyzing the transfer of a malonyl moiety from malonyl-CoA to holo acyl carrier protein (ACP), generating malonyl-ACP and free CoASH. Malonyl-ACP, which is the product of this reaction, is the key building block for de novo fatty-acid biosynthesis. Various binary complex structures of the Escherichia coli enzyme are presented, including that of the natural substrate malonyl-CoA, indicating the functional role of the highly conserved amino acids Gln11, Ser92, Arg117 and His201 and the stabilizing function of the preformed oxyanion hole during the enzymatic reaction. Based on the presented structural data, a possible new catalytic enzyme mechanism is discussed. The data obtained could be used in aiding the process of rational inhibitor design.

Characterization and inhibitor discovery of one novel malonyl-CoA: Acyl carrier protein transacylase (MCAT) from Helicobacter pylori

Type II fatty acid synthesis (FAS II) is an essential process for bacteria survival, and malonyl-CoA:acyl carrier protein transacylase (MCAT) is a key enzyme in FAS II pathway, which is responsible for transferring the malonyl group from malonyl-CoA to the holo-ACP by forming malonyl-ACP. In this work, we described the cloning, characterization and enzymatic inhibition of a new MCAT from Helicobacter pylori strain SS1 ( HpMCAT), and the gene sequence of HpfabD was deposited in the GenBank database (Accession No. AY738332). Enzymatic characterization of HpMCAT showed that the K m value for malonyl-CoA was 21.01 ± 2.3 μM, and the thermal- and guanidinium hydrochloride-induced unfolding processes for HpMCAT were quantitatively investigated by circular dichroism spectral analyses. Moreover, a natural product, corytuberine, was discovered to demonstrate inhibitory activity against HpMCAT with IC 50 value at 33.1 ± 3.29 μM. Further enzymatic assay results indicated that corytuberine inhibits HpMCAT in an uncompetitive manner. To our knowledge, this is the firstly reported MCAT inhibitor to date. This current work is hoped to supply useful information for better understanding the MCAT features of H. pylori strain, and corytuberine might be used as a potential lead compound in the discovery of the antibacterial agents using HpMCAT as target.

Ketosynthases in the initiation and elongation modules of aromatic polyketide synthases have orthogonal acyl carrier protein specificity.

Many bacterial aromatic polyketides are synthesized by type II polyketide synthases (PKSs) which minimally consist of a ketosynthase-chain length factor (KS-CLF) heterodimer, an acyl carrier protein (ACP), and a malonyl-CoA:ACP transacylase (MAT). This minimal PKS initiates polyketide biosynthesis by decarboxylation of malonyl-ACP, which is catalyzed by the KS-CLF complex and leads to incorporation of an acetate starter unit. In non-acetate-primed PKSs, such as the frenolicin (fren) PKS and the R1128 PKS, decarboxylative priming is suppressed in favor of chain initiation with alternative acyl groups. Elucidation of these unusual priming pathways could lead to the engineered biosynthesis of polyketides containing novel starter units. Unique to some non-acetate-primed PKSs is a second catalytic module comprised of a dedicated homodimeric KS, an additional ACP, and a MAT. This initiation module is responsible for starter-unit selection and catalysis of the first chain elongation step. To elucidate the protein-protein recognition features of this dissociated multimodular PKS system, we expressed and purified two priming and two elongation KSs, a set of six ACPs from diverse sources, and a MAT. In the presence of the MAT, each ACP was labeled with malonyl-CoA rapidly. In the presence of a KS-CLF and MAT, all ACPs from minimal PKSs supported polyketide synthesis at comparable rates (k(cat) between 0.17 and 0.37 min(-1)), whereas PKS activity was attenuated by at least 50-fold in the presence of an ACP from an initiation module. In contrast, the opposite specificity pattern was observed with priming KSs: while ACPs from initiation modules were good substrates, ACPs from minimal PKSs were significantly poorer substrates. Our results show that KS-CLF and KSIII recognize orthogonal sets of ACPs, and the additional ACP is indispensable for the incorporation of non-acetate primer units. Sequence alignments of the two classes of ACPs identified a tyrosine residue that is unique to priming ACPs. Site-directed mutagenesis of this amino acid in the initiation and elongation module ACPs of the R1128 PKS confirmed the importance of this residue in modulating interactions between KSs and ACPs. Our study provides new biochemical insights into unusual chain initiation mechanisms of bacterial aromatic PKSs.

Malonyl-CoA:ACP transacylase from Streptomyces coelicolor has two alternative catalytically active nucleophiles.

Fatty acids and polyketides are synthesized by mechanistically and evolutionarily related multienzyme systems. Their carbon chain backbones are synthesized via repeated decarboxylative condensations of alpha-carboxylated building blocks onto a growing acyl chain. These alpha-carboxylated building blocks are transferred from the corresponding coenzyme A thioesters onto the phosphopantetheine arm of an acyl carrier protein (ACP) by acyl transferases, which operate by a ping-pong mechanism involving an acyl-O-serine intermediate. In the course of our studies on the malonyl-CoA:ACP transacylase (MAT) from Streptomyces coelicolor, we observed that an active-site Ser (97) --> Ala mutant retains activity as well as the ability to be covalently labeled by (14)C malonyl-CoA. Here we demonstrate that an alternative, catalytically competent nucleophile exists in the active site of this enzyme. Next to the active-site serine is a histidine residue that is conserved in some, but not all acyl transferases. The H96A mutant is also active and can be labeled, but an H96A/S97A double mutant is inactive and cannot be labeled. The ability of H96 to form a malonyl-imidazole adduct was confirmed by proteolysis, followed by radio-HPLC and mass spectrometric analysis of the S97A mutant enzyme. Kinetic analysis revealed that the k(cat) of the S97A mutant was within 10-fold that of the wild-type enzyme, whereas the K(M)s of the two enzymes were comparable. Sequence comparison with the E. coli MAT (whose X-ray structure is known) led to the identification of H201 as the putative base in the serine-histidine catalytic dyad of the S. coelicolor enzyme. The absence of MAT activity in the H201A mutant and the detection of weak activity in the H201Q mutant was consistent with this proposal. The implications of this unexpected finding are discussed.

Kinetic Analysis of the Actinorhodin Aromatic Polyketide Synthase

Type II polyketide synthases (PKSs) are bacterial multienzyme systems that catalyze the biosynthesis of a broad range of natural products. A core set of subunits, consisting of a ketosynthase, a chain length factor, an acyl carrier protein (ACP) and possibly a malonyl CoA:ACP transacylase (MAT) forms a "minimal" PKS. They generate a poly-beta-ketone backbone of a specified length from malonyl-CoA derived building blocks. Here we (a) report on the kinetic properties of the actinorhodin minimal PKS, and (b) present further data in support of the requirement of the MAT. Kinetic analysis showed that the apoACP is a competitive inhibitor of minimal PKS activity, demonstrating the importance of protein-protein interactions between the polypeptide moiety of the ACP and the remainder of the minimal PKS. In further support of the requirement of MAT for PKS activity, two new findings are presented. First, we observe hyperbolic dependence of PKS activity on MAT concentration, saturating at very low amounts (half-maximal rate at 19.7 +/- 5.1 nM). Since MAT can support PKS activity at less than 1/100 the typical concentration of the ACP and ketosynthase/chain length factor components, it is difficult to rule out the presence of trace quantities of MAT in a PKS reaction mixture. Second, an S97A mutant was constructed at the nucleophilic active site of the MAT. Not only can this mutant protein support PKS activity, it is also covalently labeled by [(14)C]malonyl-CoA, demonstrating that the serine nucleophile (which has been the target of PMSF inhibition in earlier studies) is dispensible for MAT activity in a Type II PKS system.