Revealing the catalytic potential of an acyl-ACP desaturase: tandem selective oxidation of saturated fatty acids.

Department of Biology, Brookhaven National Laboratory, 50 Bell Avenue, Upton, NY 11973, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 10/2008; 105(38):14738-43. DOI: 10.1073/pnas.0805645105
Source: PubMed

ABSTRACT It is estimated that plants contain thousands of fatty acid structures, many of which arise by the action of membrane-bound desaturases and desaturase-like enzymes. The details of "unusual" e.g., hydroxyl or conjugated, fatty acid formation remain elusive, because these enzymes await structural characterization. However, soluble plant acyl-ACP (acyl carrier protein) desaturases have been studied in far greater detail but typically only catalyze desaturation (dehydrogenation) reactions. We describe a mutant of the castor acyl-ACP desaturase (T117R/G188L/D280K) that converts stearoyl-ACP into the allylic alcohol trans-isomer (E)-10-18:1-9-OH via a cis isomer (Z)-9-18:1 intermediate. The use of regiospecifically deuterated substrates shows that the conversion of (Z)-9-18:1 substrate to (E)-10-18:1-9-OH product proceeds via hydrogen abstraction at C-11 and highly regioselective hydroxylation (>97%) at C-9. (18)O-labeling studies show that the hydroxyl oxygen in the reaction product is exclusively derived from molecular oxygen. The mutant enzyme converts (E)-9-18:1-ACP into two major products, (Z)-10-18:1-9-OH and the conjugated linolenic acid isomer, (E)-9-(Z)-11-18:2. The observed product profiles can be rationalized by differences in substrate binding as dictated by the curvature of substrate channel at the active site. That three amino acid substitutions, remote from the diiron active site, expand the range of reaction outcomes to mimic some of those associated with the membrane-bound desaturase family underscores the latent potential of O(2)-dependent nonheme diiron enzymes to mediate a diversity of functionalization chemistry. In summary, this study contributes detailed mechanistic insights into factors that govern the highly selective production of unusual fatty acids.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Mycobacterium tuberculosis is a causative agent of tuberculosis. The ability of Mycobacterium tuberculosis to be quiescent in the cell has caused the emergence of latent infection. A comprehensive proteomic analysis of Mycobacterium tuberculosis H37Rv over three growth phases, namely mid-log (14-day culture), early-stationary (28-day culture) and late-stationary (50-day culture) was performed in order to study the change in proteome from the mid-log phase to late-stationary phase. Combination methods of two-dimensional electrophoresis (2-DE) and tandem mass spectrometry were used to generate proteome maps of Mycobacterium tuberculosis at different growth phases. Ten proteins were detected differentially expressed in the late-stationary phase compared to the other two phases. These proteins were SucD, TrpD and Rv2161c, which belong to metabolic pathways proteins; FadE5, AccD5, DesA1 and Rv1139c are proteins involved in cell wall or lipid biosynthesis; while TB21.7 and Rv3224 are conserved hypothetical proteins with unknown function. A surface antigen protein, DesA1 was not detectable in the late-stationary phase although present in both log and early-stationary phases. The changes in the expression levels of these proteins were in line with the growth environment changes of the bacteria from mid-log phase to late-stationary phase. The information gathered may be valuable in the intervention against latent TB infection. This article is protected by copyright. All rights reserved.
    Biotechnology and Applied Biochemistry 03/2014; 61(2). DOI:10.1002/bab.1137 · 1.32 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The hydroxylation of alkanes by heme FeIVO species occurs via the hydrogen abstraction-oxygen rebound mechanism. It has been assumed that nonheme FeIVO species follow the heme FeIVO paradigm in the C-H bond activation reactions. Herein, we report theoretical and experimental evidence that C-H bond activation of alkanes by synthetic nonheme FeIVO complexes follows an alternative mechanism. Theoretical calculations predict that dissociation of the substrate radical, which is formed past hydrogen abstraction from the alkane, is more favorable than the oxygen rebound or desaturation processes. This theoretical prediction is verified by experimental results that are obtained by analyzing iron and organic products formed in the C-H bond activation of substrates by nonheme FeIVO complexes. The different behavior between heme and nonheme FeIVO species is ascribed to differences in structural preference and exchange enhanced reactivity. Thus, the general consensus that the C-H bond activation by high-valent metal-oxo species, including nonheme FeIVO, occurs via the conventional hydrogen abstraction-oxygen rebound mechanism should be viewed with caution.
    Journal of the American Chemical Society 12/2012; 134(50). DOI:10.1021/ja308290r · 11.44 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Society has come to rely heavily on mineral oil for both energy and petrochemical needs. Plant lipids are uniquely suited to serve as a renewable source of high-value fatty acids for use as chemical feedstocks and as a substitute for current petrochemicals. Despite the broad variety of acyl structures encountered in nature and the cloning of many genes involved in their biosynthesis, attempts at engineering economic levels of specialty industrial fatty acids in major oilseed crops have so far met with only limited success. Much of the progress has been hampered by an incomplete knowledge of the fatty acid biosynthesis and accumulation pathways. This review covers new insights based on metabolic flux and reverse engineering studies that have changed our view of plant oil synthesis from a mostly linear process to instead an intricate network with acyl fluxes differing between plant species. These insights are leading to new strategies for high-level production of industrial fatty acids and waxes. Furthermore, progress in increasing the levels of oil and wax structures in storage and vegetative tissues has the potential to yield novel lipid production platforms. The challenge and opportunity for the next decade will be to marry these technologies when engineering current and new crops for the sustainable production of oil and wax feedstocks.
    Plant Biotechnology Journal 11/2012; 11(2). DOI:10.1111/pbi.12023 · 5.68 Impact Factor


Available from