Dataset Information

Allosteric modulation of the substrate specificity of acyl-CoA wax alcohol acyltransferase 2.

ABSTRACT: The esterification of alcohols with fatty acids is a universal mechanism to form inert storage forms of sterols, di- and triacylglycerols, and retinoids. In ocular tissues, formation of retinyl esters is an essential step in the enzymatic regeneration of the visual chromophore (11-cis-retinal). Acyl-CoA wax alcohol acyltransferase 2 (AWAT2), also known as multifunctional O-acyltransferase (MFAT), is an integral membrane enzyme with a broad substrate specificity that has been shown to preferentially esterify 11-cis-retinol and thus contribute to formation of a readily available pool of cis retinoids in the eye. However, the mechanism by which this promiscuous enzyme can gain substrate specificity is unknown. Here, we provide evidence for an allosteric modulation of the enzymatic activity by 11-cis retinoids. This regulation is independent from cellular retinaldehyde-binding protein (CRALBP), the major cis-retinoid binding protein. This positive-feedback regulation leads to decreased esterification rates for 9-cis, 13-cis, or all-trans retinols and thus enables preferential synthesis of 11-cis-retinyl esters. Finally, electron microscopy analyses of the purified enzyme indicate that this allosteric effect does not result from formation of functional oligomers. Altogether, these data provide the experimental basis for understanding regulation of AWAT2 substrate specificity.

SUBMITTER: Arne JM

PROVIDER: S-EPMC5392747 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Allosteric modulation of the substrate specificity of acyl-CoA wax alcohol acyltransferase 2.

Arne Jason M JM Widjaja-Adhi Made Airanthi K MAK Hughes Taylor T Huynh Kevin W KW Silvaroli Josie A JA Chelstowska Sylwia S Moiseenkova-Bell Vera Y VY Golczak Marcin M

Journal of lipid research 20170117 4

The esterification of alcohols with fatty acids is a universal mechanism to form inert storage forms of sterols, di- and triacylglycerols, and retinoids. In ocular tissues, formation of retinyl esters is an essential step in the enzymatic regeneration of the visual chromophore (11-<i>cis</i>-retinal). Acyl-CoA wax alcohol acyltransferase 2 (AWAT2), also known as multifunctional <i>O</i>-acyltransferase (MFAT), is an integral membrane enzyme with a broad substrate specificity that has been shown ...[more]

PMID: 28096191

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Project description:BACKGROUND:Triacylglycerols (TAGs) and wax esters (WEs) are important neutral lipids which serve as energy reservoir in some plants and microorganisms. In recent years, these biologically produced neutral lipids have been regarded as potential alternative energy sources for biofuel production because of the increased interest on developing renewable and environmentally benign alternatives for fossil fuels. In bacteria, the final step in TAG and WE biosynthetic pathway is catalyzed by wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT). This bifunctional WS/DGAT enzyme is also a key enzyme in biotechnological production of liquid WE via engineering of plants and microorganisms. To date, knowledge about this class of biologically and biotechnologically important enzymes is mainly from biochemical characterization of WS/DGATs from Arabidopsis, jojoba and some bacteria that can synthesize both TAGs and WEs intracellularly, whereas little is known about WS/DGATs from eukaryotic microorganisms. RESULTS:Here, we report the identification and characterization of two bifunctional WS/DGAT enzymes (designated TrWSD4 and TrWSD5) from the marine protist Thraustochytrium roseum. Both TrWSD4 and TrWSD5 comprise a WS-like acyl-CoA acyltransferase domain and the recombinant proteins purified from Escherichia coli Rosetta (DE3) have substantial WS and lower DGAT activity. They exhibit WS activity towards various-chain-length saturated and polyunsaturated acyl-CoAs and fatty alcohols ranging from C10 to C18. TrWSD4 displays WS activity with the lowest Km value of 0.14 ?M and the highest kcat/Km value of 1.46 × 105 M-1 s-1 for lauroyl-CoA (C12:0) in the presence of 100 ?M hexadecanol, while TrWSD5 exhibits WS activity with the lowest Km value of 0.96 ?M and the highest kcat/Km value of 9.83 × 104 M-1 s-1 for decanoyl-CoA (C10:0) under the same reaction condition. Both WS/DGAT enzymes have the highest WS activity at 37 and 47 °C, and WS activity was greatly decreased when temperature exceeds 47 °C. TrWSD4 and TrWSD5 are insensitive to ionic strength and reduced WS activity was observed when salt concentration exceeded 800 mM. The potential of T. roseum WS/DGATs to establish novel process for biotechnological production of WEs was demonstrated by heterologous expression in recombinant yeast. Expression of either TrWSD4 or TrWSD5 in Saccharomyces cerevisiae quadruple mutant H1246, which is devoid of storage lipids, resulted in the accumulation of WEs, but not any detectable TAGs, indicating a predominant WS activity in yeast. CONCLUSIONS:This study demonstrates both in vitro WS and DGAT activity of two T. roseum WS/DGATs, which were characterized as unspecific acyltransferases accepting a broad range of acyl-CoAs and fatty alcohols as substrates for WS activity but displaying substrate preference for medium-chain acyl-CoAs. In vivo characterization shows that these two WS/DGATs predominantly function as wax synthase and presents the feasibility for production of WEs by heterologous hosts.

Dataset Information

Allosteric modulation of the substrate specificity of acyl-CoA wax alcohol acyltransferase 2.

Publications

Allosteric modulation of the substrate specificity of acyl-CoA wax alcohol acyltransferase 2.

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