Investigations of the Structure of 3‐Methylcrotonyl‐CoA Carboxylase from <i>Achromobacter</i>

Schiele, Ulrich; Niedermeier, R. P.; Stürzer, Magdalena; Lynen, Feodor

doi:10.1111/j.1432-1033.1975.tb20998.x

Cited by 38 publications

(15 citation statements)

References 19 publications

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“…Precedence for a mechanism in which substrateacylated CoA can substitute for a substrate-acylated enzyme has been described for citrate lyase from Klebsiella aerogenes (52). Similarily, it has been shown for two biotin-dependent multisubunit enzymes, glutaconyl-CoA decarboxylase and 3-methylcrotonyl-CoA carboxylase, that the carboxytransferase subunit will react either with biotin carrier subunit or with free biotin (53)(54)(55). In C130A PHA synthase, the greatly reduced rate could result from inappropriate positioning of the 3-hydroxyl group of the bound HB-CoA for deprotonation and nucleophilic attack on the growing strand.…”

Section: Discussionmentioning

confidence: 94%

PHA Synthase from Chromatium vinosum: Cysteine 149 Is Involved in Covalent Catalysis

et al. 1998

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Polyhydroxyalkanoate synthase (PHA) from Chromatium Vinosum catalyzes the conversion of 3-hydroxybutyryl-CoA (HB-CoA) to polyhydroxybutyrate (PHB) and CoA. The synthase is composed of a ∼1:1 mixture of two subunits, PhaC and PhaE. Size-exclusion chromatography indicates that in solution PhaC and PhaE exist as large molecular weight aggregates. The holo-enzyme, PhaEC, has a specific activity of 150 units/mg. Each subunit was cloned, expressed, and purified as a (His) 6 -tagged construct. The PhaC-(His) 6 protein catalyzed polymerization with a specific activity of 0.9 unit/mg; the PhaE-(His) 6 protein was inactive (specific activity <0.001 unit/mg). Addition of PhaE-(His) 6 to PhaC-(His) 6 increased the activity several 100-fold. To investigate the priming step of the polymerization process, the PhaEC was incubated with a trimer of HB-CoA in which the terminal hydroxyl was replaced with tritium ( Sequencing by ion trap mass spectrometry showed that they were identical and that they each contained an altered cysteine (C149). One peptide contained the [ 3 H]-sT while the other two contained, in addition to the [ 3 H]-sT, one and two additional monomeric HBs, respectively. Mutation of C149 to alanine gave inactive synthase. The remaining two cysteines of PhaC, 292 and 130, were also mutated to alanine. The former had wild-type (wt) activity, while the latter had 0.004 wt % activity and was capable of making polymer. A mechanism is proposed in which PhaC contains all the elements essential for catalysis and the polymerization proceeds by covalent catalysis using C149 and potentially C130.Polyhydroxyalkanoates (PHAs 1 ) are polyoxoesters with properties that range from elastomers to thermoplastics (1-4). They are produced by a wide range of bacteria when they are placed in an environment of nutrient limitation (5). Copolymers of polyhydroxybutyrate (PHB) and polyhydroxyvalerate in the correct ratio have properties similar to the petrochemically based polypropylenes, the major component of bulk plastics (6). In 1997, the US market for thermoplastics was on the order of 40 million tons per year (7). PHAs have recently received much attention because they are biodegradable and can be generated from biorenewable sources: bacteria and plants (8). The major focus of many investigators is to make their production economically competitive with the polypropylenes. To achieve this goal, the requirements for the polymerization process need to be established. This paper focuses on the PHA synthase from Chromatium Vinosum which catalyzes the formation of PHB from 3-hydroxybutyryl-CoA (HB-CoA). Evidence is presented that two cysteines and covalent catalysis play an important role in the initiation and elongation of the polymerization process.PHA synthases from 20 organisms have now been identified. They have been divided into three classes based on their substrate specificity and subunit composition (9). The class I synthases, with the Ralstonia eutropha synthase as a prototype, are composed of a single polypeptide (∼65 k...

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Section: Discussionmentioning

confidence: 94%

PHA Synthase from Chromatium vinosum: Cysteine 149 Is Involved in Covalent Catalysis

et al. 1998

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“…All MCCases investigated to date (from bacterial, plant, and animal sources) are composed of two subunits, a larger biotinylated subunit of about 80 kD (MCC-A) and a smaller non-biotinylated subunit of about 60 kD (MCC-B; Schiele et al, 1975;Lau et al, 1980;Wurtele and Nikolau, 2000). However, two types of MCCase differing in their subunit stoichiometries and hence molecular weights have been reported.…”

Section: The Effect Of Biotin On Mccase Subunit Stoichiometrymentioning

confidence: 99%

“…The MCCase from animals (Lau et al, 1980), carrot (Daucus carota;Chen et al, 1993), maize (Zea mays; Diez et al, 1994), soybean (Glycine max; Song, 1993), and tomato (Lycopersicon esculentum; Wang, 1993) appear to have an A 6 B 6 quaternary structure, with a molecular mass of about 850 kD. In contrast, MCCase from bacteria (Schiele et al, 1975), pea (Pisum sativum), and potato (Solanum tuberosum; Alban et al, 1993) reporter transgene. Seedlings were grown in the absence of exogenous biotin to the indicated DAP.…”

Section: The Effect Of Biotin On Mccase Subunit Stoichiometrymentioning

confidence: 99%

The Role of Biotin in Regulating 3-Methylcrotonyl-Coenzyme A Carboxylase Expression in Arabidopsis

et al. 2003

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As a catalytic cofactor, biotin has a critical role in the enzymological mechanism of a number of enzymes that are essential in both catabolic and anabolic metabolic processes. In this study we demonstrate that biotin has additional non-catalytic functions in regulating gene expression in plants, which are biotin autotrophic organisms. Biotin controls expression of the biotin-containing enzyme, methylcrotonyl-coenzyme A (CoA) carboxylase by modulating the transcriptional, translational and/or posttranslational regulation of the expression of this enzyme. The bio1 mutant of Arabidopsis, which is blocked in the de novo biosynthesis of biotin, was used to experimentally alter the biotin status of this organism. In response to the bio1-associated depletion of biotin, the normally biotinylated A-subunit of methylcrotonyl-CoA carboxylase (MCCase) accumulates in its inactive apo-form, and both MCCase subunits hyperaccumulate. This hyperaccumulation occurs because the translation of each subunit mRNA is enhanced and/or because the each protein subunit becomes more stable. In addition, biotin affects the accumulation of distinct charge isoforms of MCCase. In contrast, in response to metabolic signals arising from the alteration in the carbon status of the organism, biotin modulates the transcription of the MCCase genes. These experiments reveal that in addition to its catalytic role as an enzyme cofactor, biotin has multiple roles in regulating gene expression.Biotin is a water-soluble vitamin biosynthesized by plants, some fungi, and most bacteria and is required by all living organisms for normal cellular functions and growth. Extensive genetic and biochemical studies of prokaryotic organisms have established that biotin is biosynthesized from pimeloyl-CoA and Ala via a four-reaction biosynthetic pathway (DeMoll, 1996). Less extensive studies indicate that plants biosynthesize biotin via an analogous pathway (Shellhammer and Meinke, 1990;Weaver et al., 1995;Patton et al., 1996;Patton et al., 1998; Alban et al., 2000). Of the four enzymes required for biotin biosynthesis, only the one catalyzing the terminal reaction has been molecularly characterized in plants. This enzyme, called biotin synthase, is encoded by the BIO2 gene of Arabidopsis and is a mitochondrial protein (Weaver et al., 1995;Patton et al., 1996; Baldet et al., 1997;Patton et al., 1998). Hence, in plants, biotin is biosynthesized in the mitochondria.Biotin acts as a coenzyme, covalently bound to a Lys residue of a group of enzymes that catalyze carboxylation, decarboxylation or transcarboxylation reactions (Moss and Lane, 1971). The reactions catalyzed by these enzymes are involved in diverse metabolic processes including lipogenesis (acetyl-CoA carboxylase [ACCase]), gluconeogenesis (pyruvate carboxylase), and amino acid metabolism (methylcrotonyl-CoA carboxylase [MCCase] and propionylCoA carboxylase). These enzymes share a common biochemical reaction mechanism, in which the biotin prosthetic group acts as an intermediate carrier of the carboxyl grou...

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“…In another experiment, we attempted to dissociate the two polypeptides by employing a method used to separate the two subunits of Achromobacter methylcrotonoyl-CoA carboxylase (Schiele et al, 1975). This method is based on the reduction of intermolecular disulfide bridges during dialysis of the protein fraction against a buffer containing 10 mM Gly/10 mM Cys at pH 9.8 under a nitrogen atmosphere.…”

Section: Structural Properties Of the Enzymementioning

confidence: 99%

Isolation and Characterization of Biotin Carboxylase from Pea Chloroplasts

et al. 1995

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Pea (Pisum sativum 1.) leaf acetyl-coenzyme A carboxylase (ACCase) exists as two structurally different forms: a major, chloroplastic, dissociable form and a minor, multifunctional enzyme form located i n the leaf epidermis. l h e dissociable form is able to carboxylate free D-biotin as an alternate substrate in place of the natural substrate, biotin carboxyl carrier protein. Here we report the purification of the biotin carboxylase component of the chloroplastic pea leaf ACCase. The purified enzyme, free from carboxyltransferase activity, is composed of two firmly bound polypeptides, one of which (38 kD) is biotinylated. I n contrast t o bacterial biotin carboxylase, which retains full activity upon removal of the biotin carboxyl carrier component, attempts to dissociate the two subunits of the plant complex led t o a complete loss of biotin carboxylase activity. Steady-state kinetic studies of the biotin carboxylase reaction reveal that addition of all substrates on the enzyme is sequential and that no product release is possible until all three substrates (MgATP, D-biotin, bicarbonate) are bound to the enzyme and all chemical processes at the active site are completed. In agreement with this mechanism, bicarbonate-dependent ATP hydrolysis by the enzyme is found to be strictly dependent on the presence of exogenous D-biotin i n the reaction medium.

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Investigations of the Structure of 3‐Methylcrotonyl‐CoA Carboxylase from Achromobacter

Cited by 38 publications

References 19 publications

PHA Synthase from Chromatium vinosum: Cysteine 149 Is Involved in Covalent Catalysis

PHA Synthase from Chromatium vinosum: Cysteine 149 Is Involved in Covalent Catalysis

The Role of Biotin in Regulating 3-Methylcrotonyl-Coenzyme A Carboxylase Expression in Arabidopsis

Isolation and Characterization of Biotin Carboxylase from Pea Chloroplasts

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