Acyl-ACP hydrolase

The termination of elongation is catalyzed by ACP-thioesterases (enzymes belonging to the class of acyl-ACP hydrolases). These enzymes hydrolyze acyl-ACP with the formation of free FA, whieh ean eross the plastid membrane to be reactivated outside the organelle [11]. 

As noted in Table II, all proplastids or chloroplasts synthesize oleic acid as their main unsaturated fatty acid from [ Cjacetate. Since Ohlrogge et al. (1979) have shown that all the ACP of a leaf cell is localized in the chloro-plast, it follows that the only site of stearyl-ACP desaturase must be the chloroplast. In addition, the highly specific acyl-ACP hydrolase must also be localized in the same organelle. The most effective substrate for acyl-ACP hydrolase is oleoyl-ACP, with stearoyl-ACP, palmitoyl-ACP, myristoyl-ACP, and lauroyl-ACP decreasing in activity in that order (Ohlrogge et al., 1978b). The consistent presence of the desaturase and the hydrolase in chloroplasts will be discussed in Section III. 

A specific acyl-ACP hydrolase which cleaves a specific acyl-ACP to a free acid as it is being synthesized. This acid may then be transported to another site, activated to the acyl-CoA derivative, and then inserted in the appropriate acceptor complex lipid. 

At present there is no evidence for a specific acyl-ACP transferase which has high specificity in transferring an acyl moiety to a suitable acceptor. This possibility was tested recently by Oo and Stumpf (1979) with extracts of developing coconut endosperm. Since this tissue synthesizes as its main fatty acid components medium-chain fatty acids (C12 and C14), extracts of this tissue could be employed to test this and other hypotheses concerning chain termination. These extracts displayed an active acyl-ACP hydrolase, but the specificity was essentially identical to that described for other tissues (Ohlrogge et al., 1978b). A specific acyl-ACP transferase for medium-chain acyl-CoAs was not observed. 

Leek extracts possess at least two acyl-acyl carrier protein (ACP) thioesterase (acyl-ACP hydrolase) activities. One is the well characterized oleoyl-ACP thioesterase (OTE) and the other is a stearoyl-ACP thioesterase (STE) with high specificity for its substrate. The relative activities of these two thioesterases differed between mesophyll and epidermis, STE has been separated from OTE and purified to near homogeneity by a seven-step procedure that included an ACP-affinity column. The purified STE was found to primarily hydrolyze stearoyl-ACP. 

The stearoyl-ACP desaturase-oleoyl-ACP hydrolase system has been examined recently in terms of the possibility that these two enzymes are directly involved in chain termination at the Cig level in plants (Ohlrogge et al., 1978b). The requirement for chain termination, namely, a high specificity for the chain length acyl moiety which is to be terminated, is met twice by... 

Hydrolase assays were conducted in storage buffer (25 mM Tris, 500 mM NaCl, 10 % v/v glycerol, pH 7.4) in the presence of GroEL. Typically, PedC (5 p,M) was incubated with acyl-ACPs (20 xM) in a 30 ttL reaction for various periods of time ranging from 0.5-60 min at 25 °C. The reaction was quenched using 0.1 % TFA, followed by ZipTip desalting for MS analysis (see Sect 2.2.15.1). 

Chapter 5 presents a simple method for making acyl-ACPs for use in PKS enzyme assays. This allows the synthesis of more realistic substrate mimics, where the full phosphopantetheine linker chain tethers the acyl chain to the ACP. Matthew went on to use these products to probe the substrate specificity of the acyl hydrolase from the pederin PKS, and demonstrate that its major housekeeping role is probably in targeting unwanted acetyl-ACP, which may be derived from acetyl-CoA during initial activation of the PKS by the promiscuous phosphopantetheine transferases. 

B. Ohlrogge, V. E. Shine [Pg.461]

Ohlrogge, J.B., Shine, W.E. and Stumpf, P.K. 1978. Fat metabolism in higher plants, characterization of plant acyl-ACP and acyl-CoA hydrolases. Arch.Biochem.Biophys., 189 382-391. 

Acyl-transferase (AT) domains have a highly conserved architecture from FASs to PKSs, consisting of a hydrolase core domain, and a smaller ferredoxin-like sub-domain. Together, these domains form an active-site channel for substrate binding [57, 70], Functionally, AT domains catalyse the transfer of acyl derivatives, most commonly malonyl or methyhnalonyl, from CoA to the phophopantetheine chain of an ACP. The catalytic mechanism involves a Ser-His dyad, with the catalytic serine located in the well conserved GHSXG-motif [71]. 

As briefly explained in Sect. 1.2.1, the -270 residue TE domain catalyses either a hydrolysis or an intermolecular cyclisation reaction to terminate the biosynthesis of polyketides and fatty acids, releasing the final prodnct from the assembly line. The TE domain exhibits an a/fi-hydrolase fold and is dimeric in modular type I PKS systems. The mechanism of both hydrolytic cleavage and macrolactonisa-tion commences with the transfer of the polyketide chain from the final ACP onto the active site serine of the TE, forming an acyl-TE intermediate. The hydrolytic... 

Scheme 6.2 Hypothetical movement of an acyl chain between a KS and the upstream ACP. Once the acyl-KS is formed, the acyl moiety can potentially be removed by de-acylation by the downstream ACP and subsequent hydrolysis by an acyl hydrolase. Alternatively, direct hydrolysis from the KS active site can occur...

The results presented thus far show that substrate specificity at the second elongation step exceeds that of acyl loading also highlight the potential problem of KS enzymatic stalling. In order to restore enzymatic activity either the acyl chain must hydrolyse off the active site Cys, or the upstream ACP must de-acylate the KS in a reversal of the initial acylation step. Once on the upstream ACP, the acyl chain may hydrolyse off the PPant arm, possibly catalysed by an acyl hydrolase (AH) domain (Scheme 6.2) [13]. In theory, both mechanisms are plausible, but there is a lack of any clear evidence in the literature for reversibility of the KS acylation step, other than the not unsurprising observation that a large excess of SNAC thiol was able to remove an acyl group from the KS active site by transthioesterification [6]. 

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