scholarly journals FadD19 of Rhodococcus rhodochrous DSM43269, a Steroid-Coenzyme A Ligase Essential for Degradation of C-24 Branched Sterol Side Chains

2011 ◽  
Vol 77 (13) ◽  
pp. 4455-4464 ◽  
Author(s):  
M. H. Wilbrink ◽  
M. Petrusma ◽  
L. Dijkhuizen ◽  
R. van der Geize
Keyword(s):  
Mutant Strain ◽  
Coenzyme A ◽  
Side Chain ◽  
Side Chains ◽  
Rhodococcus Rhodochrous ◽  
Content Type ◽  
Coa Ligase ◽  
Coenzyme A Ligase ◽  
Sequential Inactivation

ABSTRACTThe actinobacterial cholesterol catabolic gene cluster contains a subset of genes that encode β-oxidation enzymes with a putative role in sterol side chain degradation. We investigated the physiological roles of several genes, i.e.,fadD17,fadD19,fadE26,fadE27, andro04690DSM43269, by gene inactivation studies in mutant strain RG32 ofRhodococcus rhodochrousDSM43269. Mutant strain RG32 is devoid of 3-ketosteroid 9α-hydroxylase (KSH) activity and was constructed following the identification, cloning, and sequential inactivation of fivekshAgene homologs in strain DSM43269. We show that mutant strain RG32 is fully blocked in steroid ring degradation but capable of selective sterol side chain degradation. Except for RG32ΔfadD19, none of the mutants constructed in RG32 revealed an aberrant phenotype on sterol side chain degradation compared to parent strain RG32. Deletion offadD19in strain RG32 completely blocked side chain degradation of C-24 branched sterols but interestingly not that of cholesterol. The additional inactivation offadD17in mutant RG32ΔfadD19also did not affect cholesterol side chain degradation. Heterologously expressed FadD19DSM43269nevertheless was active toward steroid-C26-oic acid substrates. Our data identified FadD19 as a steroid-coenzyme A (CoA) ligase with an essentialin vivorole in the degradation of the side chains of C-24 branched-chain sterols. This paper reports the identification and characterization of a CoA ligase with anin vivorole in sterol side chain degradation. The high similarity (67%) between the FadD19DSM43269and FadD19H37Rvenzymes further suggests that FadD19H37Rvhas anin vivorole in sterol metabolism ofMycobacterium tuberculosisH37Rv.

scholarly journals A Novel Steroid-Coenzyme A Ligase from Novosphingobium sp. Strain Chol11 Is Essential for an Alternative Degradation Pathway for Bile Salts

2017 ◽  
Vol 84 (1) ◽  
Author(s):  
Onur Yücel ◽  
Johannes Holert ◽  
Kevin Christopher Ludwig ◽  
Sven Thierbach ◽  
Bodo Philipp
Keyword(s):  
Bile Salts ◽  
Coenzyme A ◽  
Alternative Pathway ◽  
Degradation Pathway ◽  
Side Chain ◽  
Side Chains ◽  
Content Type ◽  
C Cycle ◽  
Degrading Bacteria ◽  
Coenzyme A Ligase

ABSTRACT Bile salts such as cholate are steroid compounds with a C5 carboxylic side chain and occur ubiquitously in vertebrates. Upon their excretion into soils and waters, bile salts can serve as growth substrates for diverse bacteria. Novosphingobium sp. strain Chol11 degrades 7-hydroxy bile salts via 3-keto-7-deoxy-Δ4,6 metabolites by the dehydration of the 7-hydroxyl group catalyzed by the 7α-hydroxysteroid dehydratase Hsh2. This reaction has not been observed in the well-studied 9-10-seco degradation pathway used by other steroid-degrading bacteria indicating that strain Chol11 uses an alternative pathway. A reciprocal BLASTp analysis showed that known side chain degradation genes from other cholate-degrading bacteria (Pseudomonas stutzeri Chol1, Comamonas testosteroni CNB-2, and Rhodococcus jostii RHA1) were not found in the genome of strain Chol11. The characterization of a transposon mutant of strain Chol11 showing altered growth with cholate identified a novel steroid-24-oyl–coenzyme A ligase named SclA. The unmarked deletion of sclA resulted in a strong growth rate decrease with cholate, while growth with steroids with C3 side chains or without side chains was not affected. Intermediates with a 7-deoxy-3-keto-Δ4,6 structure, such as 3,12-dioxo-4,6-choldienoic acid (DOCDA), were shown to be likely physiological substrates of SclA. Furthermore, a novel coenzyme A (CoA)-dependent DOCDA degradation metabolite with an additional double bond in the side chain was identified. These results support the hypothesis that Novosphingobium sp. strain Chol11 harbors an alternative pathway for cholate degradation, in which side chain degradation is initiated by the CoA ligase SclA and proceeds via reaction steps catalyzed by so-far-unknown enzymes different from those of other steroid-degrading bacteria. IMPORTANCE This study provides further evidence of the diversity of metabolic pathways for the degradation of steroid compounds in environmental bacteria. The knowledge about these pathways contributes to the understanding of the CO2-releasing part of the global C cycle. Furthermore, it is useful for investigating the fate of pharmaceutical steroids in the environment, some of which may act as endocrine disruptors.

scholarly journals Pseudomonas aeruginosa PqsA Is an Anthranilate-Coenzyme A Ligase

2007 ◽  
Vol 190 (4) ◽  
pp. 1247-1255 ◽  
Author(s):  
James P. Coleman ◽  
L. Lynn Hudson ◽  
Susan L. McKnight ◽  
John M. Farrow ◽  
M. Worth Calfee ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Coenzyme A ◽  
Intercellular Signaling ◽  
Signaling Systems ◽  
Coa Ligase ◽  
Coenzyme A Ligase ◽  
Opportunistic Human Pathogen ◽  
Acyl Coenzyme A

ABSTRACT Pseudomonas aeruginosa is an opportunistic human pathogen which relies on several intercellular signaling systems for optimum population density-dependent regulation of virulence genes. The Pseudomonas quinolone signal (PQS) is a 3-hydroxy-4-quinolone with a 2-alkyl substitution which is synthesized by the condensation of anthranilic acid with a 3-keto-fatty acid. The pqsABCDE operon has been identified as being necessary for PQS production, and the pqsA gene encodes a predicted protein with homology to acyl coenzyme A (acyl-CoA) ligases. In order to elucidate the first step of the 4-quinolone synthesis pathway in P. aeruginosa, we have characterized the function of the pqsA gene product. Extracts prepared from Escherichia coli expressing PqsA were shown to catalyze the formation of anthraniloyl-CoA from anthranilate, ATP, and CoA. The PqsA protein was purified as a recombinant His-tagged polypeptide, and this protein was shown to have anthranilate-CoA ligase activity. The enzyme was active on a variety of aromatic substrates, including benzoate and chloro and fluoro derivatives of anthranilate. Inhibition of PQS formation in vivo was observed for the chloro- and fluoroanthranilate derivatives, as well as for several analogs which were not PqsA enzymatic substrates. These results indicate that the PqsA protein is responsible for priming anthranilate for entry into the PQS biosynthetic pathway and that this enzyme may serve as a useful in vitro indicator for potential agents to disrupt quinolone signaling in P. aeruginosa.

scholarly journals Novel Characteristics of Succinate Coenzyme A (Succinate-CoA) Ligases: Conversion of Malate to Malyl-CoA and CoA-Thioester Formation of Succinate AnaloguesIn Vitro

2013 ◽  
Vol 80 (1) ◽  
pp. 166-176 ◽  
Author(s):  
Johannes Christoph Nolte ◽  
Marc Schürmann ◽  
Catherine-Louise Schepers ◽  
Elvira Vogel ◽  
Jan Hendrik Wübbeler ◽  
...  
Keyword(s):  
Coenzyme A ◽  
Degradation Pathway ◽  
Ionization Mass ◽  
Content Type ◽  
E Coli ◽  
Coa Ligase ◽  
Strong Resemblance ◽  
Coa Thioesters

ABSTRACTThree succinate coenzyme A (succinate-CoA) ligases (SucCD) fromEscherichia coli,Advenella mimigardefordensisDPN7T, andAlcanivorax borkumensisSK2 were characterized regarding their substrate specificity concerning succinate analogues. Previous studies had suggested that SucCD enzymes might be promiscuous toward succinate analogues, such as itaconate and 3-sulfinopropionate (3SP). The latter is an intermediate of the degradation pathway of 3,3′-dithiodipropionate (DTDP), a precursor for the biotechnical production of polythioesters (PTEs) in bacteria. ThesucCDgenes were expressed inE. coliBL21(DE3)/pLysS. The SucCD enzymes ofE. coliandA. mimigardefordensisDPN7Twere purified in the native state using stepwise purification protocols, while SucCD fromA. borkumensisSK2 was equipped with a C-terminal hexahistidine tag at the SucD subunit. Besides the preference for the physiological substrates succinate, itaconate, ATP, and CoA, high enzyme activity was additionally determined for both enantiomeric forms of malate, amounting to 10 to 21% of the activity with succinate.Kmvalues ranged from 2.5 to 3.6 mM forl-malate and from 3.6 to 4.2 mM ford-malate for the SucCD enzymes investigated in this study. Asl-malate-CoA ligase is present in the serine cycle for assimilation of C1compounds in methylotrophs, structural comparison of these two enzymes as members of the same subsubclass suggested a strong resemblance of SucCD tol-malate-CoA ligase and gave rise to the speculation that malate-CoA ligases and succinate-CoA ligases have the same evolutionary origin. Although enzyme activities were very low for the additional substrates investigated, liquid chromatography/electrospray ionization-mass spectrometry analyses proved the ability of SucCD enzymes to form CoA-thioesters of adipate, glutarate, and fumarate. Since all SucCD enzymes were able to activate 3SP to 3SP-CoA, we consequently demonstrated that the activation of 3SP is not a unique characteristic of the SucCD fromA. mimigardefordensisDPN7T. The essential role ofsucCDin the activation of 3SPin vivowas proved by genetic complementation.

scholarly journals Characterization of Novel Acyl Coenzyme A Dehydrogenases Involved in Bacterial Steroid Degradation

2015 ◽  
Vol 197 (8) ◽  
pp. 1360-1367 ◽  
Author(s):  
Amanda Ruprecht ◽  
Jaymie Maddox ◽  
Alexander J. Stirling ◽  
Nicole Visaggio ◽  
Stephen Y. K. Seah
Keyword(s):  
Active Site ◽  
Coenzyme A ◽  
Side Chain ◽  
Side Chains ◽  
Content Type ◽  
Carbon Side Chain ◽  
Acyl Coa Dehydrogenases ◽  
Acyl Coenzyme A ◽  
Rhodococcus Jostii

ABSTRACTThe acyl coenzyme A (acyl-CoA) dehydrogenases (ACADs) FadE34 and CasC, encoded by the cholesterol and cholate gene clusters ofMycobacterium tuberculosisandRhodococcus jostiiRHA1, respectively, were successfully purified. Both enzymes differ from previously characterized ACADs in that they contain two fused acyl-CoA dehydrogenase domains in a single polypeptide. Site-specific mutagenesis showed that only the C-terminal ACAD domain contains the catalytic glutamate base required for enzyme activity, while the N-terminal ACAD domain contains an arginine required for ionic interactions with the pyrophosphate of the flavin adenine dinucleotide (FAD) cofactor. Therefore, the two ACAD domains must associate to form a single active site. FadE34 and CasC were not active toward the 3-carbon side chain steroid metabolite 3-oxo-23,24-bisnorchol-4-en-22-oyl-CoA (4BNC-CoA) but were active toward steroid CoA esters containing 5-carbon side chains. CasC has similar specificity constants for cholyl-CoA, deoxycholyl-CoA, and 3β-hydroxy-5-cholen-24-oyl-CoA, while FadE34 has a preference for the last compound, which has a ring structure similar to that of cholesterol metabolites. Knockout of thecasCgene inR. jostiiRHA1 resulted in a reduced growth on cholate as a sole carbon source and accumulation of a 5-carbon side chain cholate metabolite. FadE34 and CasC represent unique members of ACADs with primary structures and substrate specificities that are distinct from those of previously characterized ACADs.IMPORTANCEWe report here the identification and characterization of acyl-CoA dehydrogenases (ACADs) involved in the metabolism of 5-carbon side chains of cholesterol and cholate. The two homologous enzymes FadE34 and CasC, fromM. tuberculosisandRhodococcus jostiiRHA1, respectively, contain two ACAD domains per polypeptide, and we show that these two domains interact to form a single active site. FadE34 and CasC are therefore representatives of a new class of ACADs with unique primary and quaternary structures. The bacterial steroid degradation pathway is important for the removal of steroid waste in the environment and for survival of the pathogenM. tuberculosiswithin host macrophages. FadE34 is a potential target for development of new antibiotics against tuberculosis.

scholarly journals Conversion of 4-Hydroxybutyrate to Acetyl Coenzyme A and Its Anapleurosis in the Metallosphaera sedula 3-Hydroxypropionate/4-Hydroxybutyrate Carbon Fixation Pathway

2014 ◽  
Vol 80 (8) ◽  
pp. 2536-2545 ◽  
Author(s):  
Aaron B. Hawkins ◽  
Michael W. W. Adams ◽  
Robert M. Kelly
Keyword(s):  
Carbon Fixation ◽  
Coenzyme A ◽  
Flux Analysis ◽  
Central Metabolism ◽  
Acetyl Coa ◽  
Acetyl Coenzyme A ◽  
Content Type ◽  
Metallosphaera Sedula ◽  
Acetyl Coenzyme ◽  
Coa Ligase

ABSTRACTThe extremely thermoacidophilic archaeonMetallosphaera sedula(optimum growth temperature, 73°C, pH 2.0) grows chemolithoautotrophically on metal sulfides or molecular hydrogen by employing the 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) carbon fixation cycle. This cycle adds two CO2molecules to acetyl coenzyme A (acetyl-CoA) to generate 4HB, which is then rearranged and cleaved to form two acetyl-CoA molecules. Previous metabolic flux analysis showed that two-thirds of central carbon precursor molecules are derived from succinyl-CoA, which is oxidized to malate and oxaloacetate. The remaining one-third is apparently derived from acetyl-CoA. As such, the steps beyond succinyl-CoA are essential for completing the carbon fixation cycle and for anapleurosis of acetyl-CoA. Here, the final four enzymes of the 3HP/4HB cycle, 4-hydroxybutyrate-CoA ligase (AMP forming) (Msed_0406), 4-hydroxybutyryl-CoA dehydratase (Msed_1321), crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase (Msed_0399), and acetoacetyl-CoA β-ketothiolase (Msed_0656), were produced recombinantly inEscherichia coli, combinedin vitro, and shown to convert 4HB to acetyl-CoA. Metabolic pathways connecting CO2fixation and central metabolism were examined using a gas-intensive bioreactor system in whichM. sedulawas grown under autotrophic (CO2-limited) and heterotrophic conditions. Transcriptomic analysis revealed the importance of the 3HP/4HB pathway in supplying acetyl-CoA to anabolic pathways generating intermediates inM. sedulametabolism. The results indicated that flux between the succinate and acetyl-CoA branches in the 3HP/4HB pathway is governed by 4-hydroxybutyrate-CoA ligase, possibly regulated posttranslationally by the protein acetyltransferase (Pat)/Sir2-dependent system. Taken together, this work confirms the final four steps of the 3HP/4HB pathway, thereby providing the framework for examining connections between CO2fixation and central metabolism inM. sedula.

scholarly journals Benzoate-Coenzyme A Ligase from Thauera aromatica: an Enzyme Acting in Anaerobic and Aerobic Pathways

2003 ◽  
Vol 185 (16) ◽  
pp. 4920-4929 ◽  
Author(s):  
Karola Schühle ◽  
Johannes Gescher ◽  
Ulrich Feil ◽  
Michael Paul ◽  
Martina Jahn ◽  
...  
Keyword(s):  
Anaerobic Metabolism ◽  
Coenzyme A ◽  
Aromatic Acids ◽  
Aerobic Growth ◽  
Aromatic Substrates ◽  
Thauera Aromatica ◽  
Coa Ligase ◽  
Anaerobic Aromatic Metabolism ◽  
Coenzyme A Ligase ◽  
Specificity Constant

ABSTRACT In the denitrifying member of the β-Proteobacteria Thauera aromatica, the anaerobic metabolism of aromatic acids such as benzoate or 2-aminobenzoate is initiated by the formation of the coenzyme A (CoA) thioester, benzoyl-CoA and 2-aminobenzoyl-CoA, respectively. Both aromatic substrates were transformed to the acyl-CoA intermediate by a single CoA ligase (AMP forming) that preferentially acted on benzoate. This benzoate-CoA ligase was purified and characterized as a 57-kDa monomeric protein. Based on V max/Km , the specificity constant for 2-aminobenzoate was 15 times lower than that for benzoate; this may be the reason for the slower growth on 2-aminobenzoate. The benzoate-CoA ligase gene was cloned and sequenced and was found not to be part of the gene cluster encoding the general benzoyl-CoA pathway of anaerobic aromatic metabolism. Rather, it was located in a cluster of genes coding for a novel aerobic benzoate oxidation pathway. In line with this finding, the same CoA ligase was induced during aerobic growth with benzoate. A deletion mutant not only was unable to grow anaerobically on benzoate or 2-aminobenzoate, but also aerobic growth on benzoate was affected. This suggests that benzoate induces a single benzoate-CoA ligase. The product of benzoate activation, benzoyl-CoA, then acts as inducer of separate anaerobic or aerobic pathways of benzoyl-CoA, depending on whether oxygen is lacking or present.

scholarly journals Coenzyme F420-Dependent Glucose-6-Phosphate Dehydrogenase-Coupled Polyglutamylation of Coenzyme F420in Mycobacteria

2018 ◽  
Vol 200 (23) ◽  
Author(s):  
Endang Purwantini ◽  
Usha Loganathan ◽  
Biswarup Mukhopadhyay
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Genetic Analysis ◽  
The Other ◽  
Side Chain ◽  
Side Chains ◽  
Content Type ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal ◽  
Glucose 6 Phosphate Dehydrogenase

ABSTRACTCoenzyme F420plays a key role in the redox metabolisms of various archaea and bacteria, includingMycobacterium tuberculosis. InM. tuberculosis, F420-dependent reactions have been linked to several virulence factors. F420carries multiple glutamate residues in the side chain, forming F420-nspecies (n, number of glutamate residues), and the length of this side chain impacts cellular physiology.M. tuberculosisstrains with F420species carrying shorter side chains exhibit resistance to delamanid and pretomanid, two new tuberculosis (TB) drugs. Thus, the process of polyglutamylation of F420is of great interest. It has been known from genetic analysis that in mycobacteria an F420-0 γ-glutamyl ligase (FbiB) introduces up to seven glutamate residues into F420. However, purified FbiB ofM. tuberculosis(MtbFbiB) is either inefficient or incapable of incorporating more than two glutamates. We found that,in vitro,MtbFbiB synthesized side chains containing up to seven glutamate residues if F420was presented to the enzyme in a two-electron reduced state (F420H2). Our genetic analysis inMycobacterium bovisBCG andMycobacterium smegmatisand an analysis of literature data onM. tuberculosisrevealed that in these mycobacteria the polyglutamylation process requires the assistance of F420-dependent glucose-6-phosphate dehydrogenase (Fgd) which reduces F420to F420H2. We hypothesize that, starting with F420-0H2, the amino-terminal domain of FbiB builds F420-2H2, which is then transferred to the carboxy-terminal domain for further glutamylation; F420-2H2modifies the carboxy-terminal domain structurally to accommodate longer glutamyl chains. This system is analogous to folylpolyglutamate synthase, which introduces more than one glutamate residue into folate only after this vitamin is reduced to tetrahydrofolate.IMPORTANCECoenzyme F420-dependent reactions ofMycobacterium tuberculosis, which causes tuberculosis, potentially contributes to the virulence of this bacterium. The coenzyme carries a glutamic acid-derived tail, the length of which influences the metabolism ofM. tuberculosis. Mutations that eliminate the production of F420with longer tails makeM. tuberculosisresistant to two new tuberculosis drugs. This report describes that the synthesis of longer glutamyl tails of F420requires concerted actions of two enzymes, one of which reduces the coenzyme prior to the action of the other, which catalyzes polyglutamylation. This knowledge will help to develop more effective tuberculosis (TB) drugs. Remarkably, the introduction of multiple glutamate residues into the sidechain of folate (vitamin B9) requires similar concerted actions, where one enzyme reduces the vitamin to tetrahydrofolate and the other catalyzes polyglutamylation; folate is required for DNA and amino acid synthesis. Thus, the reported research has also revealed a key similarity between two important cellular systems.

scholarly journals Peroxisomes Are Required for Efficient Penicillin Biosynthesis in Penicillium chrysogenum

2010 ◽  
Vol 76 (17) ◽  
pp. 5702-5709 ◽  
Author(s):  
Wiebe H. Meijer ◽  
Loknath Gidijala ◽  
Susan Fekken ◽  
Jan A. K. W. Kiel ◽  
Marco A. van den Berg ◽  
...  
Keyword(s):  
Penicillium Chrysogenum ◽  
Coenzyme A ◽  
Antibiotic Production ◽  
Hyphal Growth ◽  
Side Chain ◽  
Penicillin Biosynthesis ◽  
Peroxisome Proliferation ◽  
Coa Ligase ◽  
Penicillanic Acid ◽  
Acyl Coenzyme A

ABSTRACT In the fungus Penicillium chrysogenum, penicillin (PEN) production is compartmentalized in the cytosol and in peroxisomes. Here we show that intact peroxisomes that contain the two final enzymes of PEN biosynthesis, acyl coenzyme A (CoA):6-amino penicillanic acid acyltransferase (AT) as well as the side-chain precursor activation enzyme phenylacetyl CoA ligase (PCL), are crucial for efficient PEN synthesis. Moreover, increasing PEN titers are associated with increasing peroxisome numbers. However, not all conditions that result in enhanced peroxisome numbers simultaneously stimulate PEN production. We find that conditions that lead to peroxisome proliferation but simultaneously interfere with the normal physiology of the cell may be detrimental to antibiotic production. We furthermore show that peroxisomes develop in germinating conidiospores from reticule-like structures. During subsequent hyphal growth, peroxisome proliferation occurs at the tip of the growing hyphae, after which the organelles are distributed over newly formed subapical cells. We observed that the organelle proliferation machinery requires the dynamin-like protein Dnm1.

scholarly journals Click Chemistry-Facilitated Structural Diversification of Nitrothiazoles, Nitrofurans, and Nitropyrroles Enhances Antimicrobial Activity against Giardia lamblia

2017 ◽  
Vol 61 (6) ◽  
Author(s):  
Wan Jung Kim ◽  
Keith A. Korthals ◽  
Suhua Li ◽  
Christine Le ◽  
Jarosław Kalisiak ◽  
...  
Keyword(s):  
Click Chemistry ◽  
Giardia Lamblia ◽  
Diarrheal Disease ◽  
Wide Spectrum ◽  
Fold Increase ◽  
Side Chain ◽  
Full Potential ◽  
Content Type

ABSTRACT Giardia lamblia is an important and ubiquitous cause of diarrheal disease. The primary agents in the treatment of giardiasis are nitroheterocyclic drugs, particularly the imidazoles metronidazole and tinidazole and the thiazole nitazoxanide. Although these drugs are generally effective, treatment failures occur in up to 20% of cases, and resistance has been demonstrated in vivo and in vitro. Prior work had suggested that side chain modifications of the imidazole core can lead to new effective 5-nitroimidazole drugs that can combat nitro drug resistance, but the full potential of nitroheterocycles other than imidazole to yield effective new antigiardial agents has not been explored. Here, we generated derivatives of two clinically utilized nitroheterocycles, nitrothiazole and nitrofuran, as well as a third heterocycle, nitropyrrole, which is related to nitroimidazole but has not been systematically investigated as an antimicrobial drug scaffold. Click chemistry was employed to synthesize 442 novel nitroheterocyclic compounds with extensive side chain modifications. Screening of this library against representative G. lamblia strains showed a wide spectrum of in vitro activities, with many of the compounds exhibiting superior activity relative to reference drugs and several showing >100-fold increase in potency and the ability to overcome existing forms of metronidazole resistance. The majority of new compounds displayed no cytotoxicity against human cells, and several compounds were orally active against murine giardiasis in vivo. These findings provide additional impetus for the systematic development of nitroheterocyclic compounds with nonimidazole cores as alternative and improved agents for the treatment of giardiasis and potentially other infectious agents.

scholarly journals Hyperglutamylation of Tubulin Can either Stabilize or Destabilize Microtubules in the Same Cell

2009 ◽  
Vol 9 (1) ◽  
pp. 184-193 ◽  
Author(s):  
Dorota Wloga ◽  
Drashti Dave ◽  
Jennifer Meagley ◽  
Krzysztof Rogowski ◽  
Maria Jerka-Dziadosz ◽  
...  
Keyword(s):  
Chain Length ◽  
Eukaryotic Cells ◽  
Side Chain ◽  
Side Chains ◽  
Primary Sequence ◽  
Side Chain Length ◽  
Length Regulation ◽  
Cytoplasmic Microtubules

ABSTRACT In most eukaryotic cells, tubulin is subjected to posttranslational glutamylation, a conserved modification of unclear function. The glutamyl side chains form as branches of the primary sequence glutamic acids in two biochemically distinct steps: initiation and elongation. The length of the glutamyl side chain is spatially controlled and microtubule type specific. Here, we probe the significance of the glutamyl side chain length regulation in vivo by overexpressing a potent side chain elongase enzyme, Ttll6Ap, in Tetrahymena. Overexpression of Ttll6Ap caused hyperelongation of glutamyl side chains on the tubulin of axonemal, cortical, and cytoplasmic microtubules. Strikingly, in the same cell, hyperelongation of glutamyl side chains stabilized cytoplasmic microtubules and destabilized axonemal microtubules. Our observations suggest that the cellular outcomes of glutamylation are mediated by spatially restricted tubulin interactors of diverse nature.

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