The Escherichia coli Malonyl-CoA:Acyl Carrier Protein Transacylase at 1.5-Å Resolution.

Abstract

Endogenous fatty acids are synthesized in all organisms in a pathway catalyzed by the fatty acid synthase complex. In bacteria, where the fatty acids are used primarily for incorporation into components of cell membranes, fatty acid synthase is made up of several independent cytoplasmic enzymes, each catalyzing one specific reaction. The initiation of the elongation step, which extends the length of the growing acyl chain by two carbons, requires the transfer of the malonyl moiety from malonyl-CoA onto the acyl carrier protein. We report here the crystal structure (refined at 1.5-A resolution to an R factor of 0.19) of the malonyl-CoA specific transferase from Escherichia coli. The protein has an alpha/beta type architecture, but its fold is unique. The active site inferred from the location of the catalytic Ser-92 contains a typical nucleophilic elbow as observed in alpha/beta hydrolases. Serine 92 is hydrogen bonded to His-201 in a fashion similar to various serine hydrolases. However, instead of a carboxyl acid typically found in catalytic triads, the main chain carbonyl of Gln-250 serves as a hydrogen bond acceptor in an interaction with His-201. Two other residues, Arg-117 and Glu-11, are also located in the active site, although their function is not clear.

Divergent sequence motifs correlated with the substrate specificity of (methyl)malonyl‐CoA:acyl carrier protein transacylase domains in modular polyketide synthases

“…MSAS represents the 6-methylsalicylic acid synthase AT [16], and MAS that of mycocerosic acid synthase [17]. The sequence of the Escherichia coli malonyl-CoA:acyl carrier protein transacylase (MCAT) [6] is also shown. Ery 0 and Ave 0 indicate AT domains that load the starter unit in the erythromycin [12] and avermectin PKSs respectively.…”

“…However, a key question that remains, even given the availability of a high resolution X-ray crystal structure for the malonyl-CoA:ACP transferase from Escherichia coli fatty acid synthase [6], is the basis for the specificity of individual acylCoA:ACP acyltransferase (AT) domains or enzymes, for the transfer of either malonyl or methylmalonyl groups onto ACE The nucleotide sequence is now available for the entire gene cluster encoding the modular polyketide synthase for biosynthesis of the immunosuppressant rapamycin, which contains seven propionate and seven acetate units [7]. Comparison of the sequences of these fourteen AT domains with the sequences of other ATs for fatty acid and polyketide synthases has been used here to identify divergent sequence motifs that clearly distinguish a malonyl-from a methylmalonyl-specific AT domain in a modular polyketide synthase.…”

“…Comparison of the sequences of these fourteen AT domains with the sequences of other ATs for fatty acid and polyketide synthases has been used here to identify divergent sequence motifs that clearly distinguish a malonyl-from a methylmalonyl-specific AT domain in a modular polyketide synthase. Although this search was conducted without reference to the possible location of the active site, a divergent sequence motif was localized in a helix which contains an invariant Gln (Gln-63 in the E. coli MCAT) that is in the active site [6]. However, the residues of the motif are too far removed to be in direct contact with the substratebinding site.…”

Asparagine and glutamine: using hydrogen atom contacts in the choice of side-chain amide orientation 1 1Edited by J. Thornton

“…Therefore, round 3 was expanded to include visual examination of all Asn/Gln/His in those two categories. Most of the resulting reassignments involve con®rming the orientation indicated by thē ip scores: for example, Gln61 of 1MLA, malonyl CoA carrier protein (Serre et al, 1995), with a score difference of 0.48 was promoted from marginal tō ipped, because it can make weak H-bonds both to backbone and to a Glu O e in the preferablē ipped orientation. However, there are a small but signi®cant number of cases where the visual assignment contradicts the direction of the score Figure 5.…”

Genetic characterization of enzymes involved in the priming steps of oxytetracycline biosynthesis in Streptomyces rimosus

“…Finally, to demonstrate that OxyP plays a catalytic role in suppressing the acetate starter unit, we performed site-directed mutagenesis of the OxyP active site. OxyP contains the signature thioesterase/acyltransferase active-site motif GxSxG, in which the side-chain hydroxyl of serine serves as the nucleophile in the proposed hydrolysis of acetyl-ACP (Serre et al, 1995). We introduced the mutant oxyP* gene, in which the active-site serine was mutated to an alanine, into the DoxyP strain WP3 using the same complementation strategy as described above to generate WP3/pDP33.…”