A Light‐Activated Acyl Carrier Protein “Trap” for Intermediate Capture in Type II Iterative Polyketide Biocatalysis

Mitochondrial LYRM (leucine/tyrosine/arginine motif) proteins are members of the Complex1_LYR-like superfamily. Individual LYRM proteins have been identified as accessory subunits or assembly factors of mitochondrial OXPHOS (oxidative phosphorylation) complexes I, II, III and V respectively, and they play particular roles in the essential Fe–S cluster biogenesis and in acetate metabolism. LYRM proteins have been implicated in mitochondrial dysfunction, e.g. in the context of insulin resistance. However, the functional significance of the common LYRM is still unknown. Analysis of protein–protein interaction screens suggests that LYRM proteins form protein complexes with phylogenetically ancient proteins of bacterial origin. Interestingly, the mitochondrial FAS (fatty acid synthesis) type II acyl-carrier protein ACPM associates with some of the LYRM protein-containing complexes. Eukaryotic LYRM proteins interfere with mitochondrial homoeostasis and might function as adaptor-like ‘accessory factors’.

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Enoyl-Acyl Carrier Protein Reductase I (FabI) Is Essential for the Intracellular Growth of Listeria monocytogenes

Infection and Immunity ◽

10.1128/iai.00647-16 ◽

2016 ◽

Vol 84 (12) ◽

pp. 3597-3607 ◽

Cited By ~ 8

Author(s):

Jiangwei Yao ◽

Megan E. Ericson ◽

Matthew W. Frank ◽

Charles O. Rock

Keyword(s):

Fatty Acids ◽

Fatty Acid ◽

Fatty Acid Synthesis ◽

Acyl Carrier Protein ◽

Acid Synthesis ◽

Carrier Protein ◽

Type Ii ◽

Intracellular Growth ◽

Content Type ◽

Bacterial Fatty Acid

Enoyl-acyl carrier protein reductase catalyzes the last step in each elongation cycle of type II bacterial fatty acid synthesis and is a key regulatory protein in bacterial fatty acid synthesis. Genes of the facultative intracellular pathogenListeria monocytogenesencode two functional enoyl-acyl carrier protein isoforms based on their ability to complement the temperature-sensitive growth phenotype ofEscherichia colistrain JP1111 [fabI(Ts)]. The FabI isoform was inactivated by the FabI selective inhibitor AFN-1252, but the FabK isoform was not affected by the drug, as expected. Inhibition of FabI by AFN-1252 decreased endogenous fatty acid synthesis by 80% and lowered the growth rate ofL. monocytogenesin laboratory medium. Robust exogenous fatty acid incorporation was not detected inL. monocytogenesunless the pathway was partially inactivated by AFN-1252 treatment. However, supplementation with exogenous fatty acids did not restore normal growth in the presence of AFN-1252. FabI inactivation prevented the intracellular growth ofL. monocytogenes, showing that neither FabK nor the incorporation of host cellular fatty acids was sufficient to support the intracellular growth ofL. monocytogenes. Our results show that FabI is the primary enoyl-acyl carrier protein reductase of type II bacterial fatty acid synthesis and is essential for the intracellular growth ofL. monocytogenes.

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Reaction mechanism of recombinant 3-oxoacyl-(acyl-carrier-protein) synthase III from Cuphea wrightii embryo, a fatty acid synthase type II condensing enzyme

Biochemical Journal ◽

10.1042/0264-6021:3450153 ◽

2000 ◽

Vol 345 (1) ◽

pp. 153 ◽

Cited By ~ 5

Author(s):

Amine ABBADI ◽

Monika BRUMMEL ◽

Burkhardt S. SCHÜTT ◽

Mary B. SLABAUGH ◽

Ricardo SCHUCH ◽

...

Keyword(s):

Fatty Acid ◽

Reaction Mechanism ◽

Fatty Acid Synthase ◽

Acyl Carrier Protein ◽

Carrier Protein ◽

Type Ii ◽

Condensing Enzyme

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The Structure of (3R)-Hydroxyacyl-Acyl Carrier Protein Dehydratase (FabZ) fromPseudomonas aeruginosa

Journal of Biological Chemistry ◽

10.1074/jbc.m408105200 ◽

2004 ◽

Vol 279 (50) ◽

pp. 52593-52602 ◽

Cited By ~ 91

Author(s):

Matthew S. Kimber ◽

Fernando Martin ◽

Yingjie Lu ◽

Simon Houston ◽

Masoud Vedadi ◽

...

Keyword(s):

Fatty Acid ◽

Fatty Acid Biosynthesis ◽

Acyl Carrier Protein ◽

Site Directed Mutagenesis ◽

Isomerization Reaction ◽

Antibacterial Drug ◽

Carrier Protein ◽

Type Ii ◽

Active Site Residues ◽

Acid Biosynthesis

Type II fatty acid biosynthesis systems are essential for membrane formation in bacteria, making the constituent proteins of this pathway attractive targets for antibacterial drug discovery. The third step in the elongation cycle of the type II fatty acid biosynthesis is catalyzed by β-hydroxyacyl-(acyl carrier protein) (ACP) dehydratase. There are two isoforms. FabZ, which catalyzes the dehydration of (3R)-hydroxyacyl-ACP totrans-2-acyl-ACP, is a universally expressed component of the bacterial type II system. FabA, the second isoform, as has more limited distribution in nature and, in addition to dehydration, also carries out the isomerization oftrans-2- tocis-3-decenoyl-ACP as an essential step in unsaturated fatty acid biosynthesis. We report the structure of FabZ from the important human pathogenPseudomonas aeruginosaat 2.5 Å of resolution.PaFabZ is a hexamer (trimer of dimers) with the His/Glu catalytic dyad located within a deep, narrow tunnel formed at the dimer interface. Site-directed mutagenesis experiments showed that the obvious differences in the active site residues that distinguish the FabA and FabZ subfamilies of dehydratases do not account for the unique ability of FabA to catalyze isomerization. Because the catalytic machinery of the two enzymes is practically indistinguishable, the structural differences observed in the shape of the substrate binding channels of FabA and FabZ lead us to hypothesize that the different shapes of the tunnels control the conformation and positioning of the bound substrate, allowing FabA, but not FabZ, to catalyze the isomerization reaction.

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Self-Malonylation Is an Intrinsic Property of a Chemically Synthesized Type II Polyketide Synthase Acyl Carrier Protein†,‡

Biochemistry ◽

10.1021/bi051499i ◽

2005 ◽

Vol 44 (46) ◽

pp. 15414-15421 ◽

Cited By ~ 34

Author(s):

Christopher J. Arthur ◽

Anna Szafranska ◽

Simon E. Evans ◽

Stuart C. Findlow ◽

Steven G. Burston ◽

...

Keyword(s):

Polyketide Synthase ◽

Acyl Carrier Protein ◽

Intrinsic Property ◽

Carrier Protein ◽

Type Ii

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Binding and pKa Modulation of a Polycyclic Substrate Analogue in a Type II Polyketide Acyl Carrier Protein

ACS Chemical Biology ◽

10.1021/cb200004k ◽

2011 ◽

Vol 6 (5) ◽

pp. 413-418 ◽

Cited By ~ 22

Author(s):

Robert W. Haushalter ◽

Fabian V. Filipp ◽

Kwang-seuk Ko ◽

Ricky Yu ◽

Stanley J. Opella ◽

...

Keyword(s):

Acyl Carrier Protein ◽

Carrier Protein ◽

Type Ii ◽

Substrate Analogue

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Depletion of Mitochondrial Acyl Carrier Protein in Bloodstream-Form Trypanosoma brucei Causes a Kinetoplast Segregation Defect

Eukaryotic Cell ◽

10.1128/ec.00290-10 ◽

2011 ◽

Vol 10 (3) ◽

pp. 286-292 ◽

Cited By ~ 17

Author(s):

April M. Clayton ◽

Jennifer L. Guler ◽

Megan L. Povelones ◽

Eva Gluenz ◽

Keith Gull ◽

...

Keyword(s):

Fatty Acid ◽

Trypanosoma Brucei ◽

Fatty Acid Synthase ◽

Acyl Carrier Protein ◽

Phospholipid Composition ◽

Conditional Knockout ◽

Bloodstream Form ◽

Carrier Protein ◽

Type Ii ◽

Kinetoplast Dna

ABSTRACT Like other eukaryotes, trypanosomes have an essential type II fatty acid synthase in their mitochondrion. We have investigated the function of this synthase in bloodstream-form parasites by studying the effect of a conditional knockout of acyl carrier protein (ACP), a key player in this fatty acid synthase pathway. We found that ACP depletion not only caused small changes in cellular phospholipids but also, surprisingly, caused changes in the kinetoplast. This structure, which contains the mitochondrial genome in the form of a giant network of several thousand interlocked DNA rings (kinetoplast DNA [kDNA]), became larger in some cells and smaller or absent in others. We observed the same pattern in isolated networks viewed by either fluorescence or electron microscopy. We found that the changes in kDNA size were not due to the disruption of replication but, instead, to a defect in segregation. kDNA segregation is mediated by the tripartite attachment complex (TAC), and we hypothesize that one of the TAC components, a differentiated region of the mitochondrial double membrane, has an altered phospholipid composition when ACP is depleted. We further speculate that this compositional change affects TAC function, and thus kDNA segregation.

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Activity Mapping the Acyl Carrier Protein - Elongating Ketosynthase Interaction in Fatty Acid Biosynthesis

10.1101/2020.07.09.196451 ◽

2020 ◽

Author(s):

Jeffrey T. Mindrebo ◽

Laetitia E. Misson ◽

Caitlin Johnson ◽

Joseph P. Noel ◽

Michael D. Burkart

Keyword(s):

Fatty Acid ◽

Active Sites ◽

Fatty Acid Biosynthesis ◽

Acyl Carrier Protein ◽

Site Directed Mutagenesis ◽

Chain Extension ◽

Carrier Protein ◽

Type Ii ◽

Multiple Partner ◽

Carbon Carbon Bond

ABSTRACTElongating ketosynthases (KSs) catalyze carbon-carbon bond forming reactions during the committed step for each round of chain extension in both fatty acid synthases (FASs) and polyketide synthases (PKSs). A small α-helical acyl carrier protein (ACP) shuttles fatty acyl intermediates between enzyme active sites. To accomplish this task, ACP relies on a series of dynamic interactions with multiple partner enzymes of FAS and associated FAS-dependent pathways. Recent structures of the Escherichia coli FAS ACP, AcpP, in covalent complexes with its two cognate elongating KSs, FabF and FabB, provide high-resolution detail of these interfaces, but a systematic analysis of specific interfacial interactions responsible for stabilizing these complexes has not yet been undertaken. Here, we use site-directed mutagenesis with both in vitro and in vivo activity analyses to quantitatively evaluate these contacting surfaces between AcpP and FabF. We delineate the FabF interface into three interacting regions and demonstrate the effects of point mutants, double mutants, and region delete variants. Results from these analyses reveal a robust and modular FabF interface capable of tolerating seemingly critical interface mutations with only the deletion of entire regions significantly compromising activity. Structure and sequence analysis of FabF orthologs from related type II FAS pathways indicate significant conservation of type II FAS KS interface residues and, overall, support its delineation into interaction regions. These findings strengthen our mechanistic understanding of molecular recognition events between ACPs and FAS enzymes and provide a blueprint for engineering ACP-dependent biosynthetic pathways.

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Suppression offabBMutation byfabF1Is Mediated by Transcription Read-through in Shewanella oneidensis

Journal of Bacteriology ◽

10.1128/jb.00463-16 ◽

2016 ◽

Vol 198 (22) ◽

pp. 3060-3069 ◽

Cited By ~ 7

Author(s):

Meng Li ◽

Qiu Meng ◽

Huihui Fu ◽

Qixia Luo ◽

Haichun Gao

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthesis ◽

Tandem Repeats ◽

Unsaturated Fatty Acids ◽

Acyl Carrier Protein ◽

Shewanella Oneidensis ◽

Acid Synthesis ◽

Carrier Protein ◽

Type Ii ◽

Content Type

ABSTRACTAs type II fatty acid synthesis is essential for the growth ofEscherichia coli, its many components are regarded as potential targets for novel antibacterial drugs. Among them, β-ketoacyl-acyl carrier protein (ACP) synthase (KAS) FabB is the exclusive factor for elongation of thecis-3-decenoyl-ACP (cis-3-C10-ACP). In our previous study, we presented evidence to suggest that this may not be the case inShewanella oneidensis, an emerging model gammaproteobacterium renowned for its respiratory versatility. Here, we identified FabF1, another KAS, as a functional replacement for FabB inS. oneidensis. InfabB+ordesA+(encoding a desaturase) cells, which are capable of making unsaturated fatty acids (UFA), FabF1 is barely produced. However, UFA auxotroph mutants devoid of bothfabBanddesAgenes can be spontaneously converted to suppressor strains, which no longer require exogenous UFAs for growth. Suppression is caused by a TGTTTT deletion in the region upstream of thefabF1gene, resulting in enhanced FabF1 production. We further demonstrated that the deletion leads to transcription read-through of the terminator foracpP, an acyl carrier protein gene immediately upstream offabF1. There are multiple tandem repeats in the region covering the terminator, and the TGTTTT deletion, as well as others, compromises the terminator efficacy. In addition, FabF2 also shows an ability to complement the FabB loss, albeit substantially less effectively than FabF1.IMPORTANCEIt has been firmly established that FabB for UFA synthesis via type II fatty acid synthesis in FabA-containing bacteria such asE. coliis essential. However,S. oneidensisappears to be an exception. In this bacterium, FabF1, when sufficiently expressed, is able to fully complement the FabB loss. Importantly, such a capability can be obtained by spontaneous mutations, which lead to transcription read-through. Therefore, our data, by identifying the functional overlap between FabB and FabFs, provide new insights into the current understanding of KAS and help reveal novel ways to block UFA synthesis for therapeutic purposes.

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Slow-tight-binding inhibition of enoyl-acyl carrier protein reductase from Plasmodium falciparum by triclosan

Biochemical Journal ◽

10.1042/bj20031821 ◽

2004 ◽

Vol 381 (3) ◽

pp. 719-724 ◽

Cited By ~ 50

Author(s):

Mili KAPOOR ◽

C. Chandramouli REDDY ◽

M. V. KRISHNASASTRY ◽

Namita SUROLIA ◽

Avadhesha SUROLIA

Keyword(s):

Plasmodium Falciparum ◽

Fatty Acid ◽

Rate Constants ◽

Enzyme Inhibitor ◽

Acyl Carrier Protein ◽

Tight Binding ◽

Carrier Protein ◽

Type I ◽

Type Ii ◽

Inhibitor Complex

Triclosan is a potent inhibitor of FabI (enoyl-ACP reductase, where ACP stands for acyl carrier protein), which catalyses the last step in a sequence of four reactions that is repeated many times with each elongation step in the type II fatty acid biosynthesis pathway. The malarial parasite Plasmodium falciparum also harbours the genes and is capable of synthesizing fatty acids by utilizing the enzymes of type II FAS (fatty acid synthase). The basic differences in the enzymes of type I FAS, present in humans, and type II FAS, present in Plasmodium, make the enzymes of this pathway a good target for antimalarials. The steady-state kinetics revealed time-dependent inhibition of FabI by triclosan, demonstrating that triclosan is a slow-tight-binding inhibitor of FabI. The inhibition followed a rapid equilibrium step to form a reversible enzyme–inhibitor complex (EI) that isomerizes to a second enzyme–inhibitor complex (EI*), which dissociates at a very slow rate. The rate constants for the isomerization of EI to EI* and the dissociation of EI* were 5.49×10−2 and 1×10−4 s−1 respectively. The Ki value for the formation of the EI complex was 53 nM and the overall inhibition constant Ki* was 96 pM. The results match well with the rate constants derived independently from fluorescence analysis of the interaction of FabI and triclosan, as well as those obtained by surface plasmon resonance studies [Kapoor, Mukhi, N. Surolia, Sugunda and A. Surolia (2004) Biochem. J. 381, 725–733].

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