Structural characterization of the entire 1.3S subunit of transcarboxylase from <i>Propionibacterium shermanii</i>

Reddy, D V S; Rothemund, Sven; Sönnichsen, Frank D.; Shenoy, Bhami C.; Carey, Paul R.

doi:10.1002/pro.5560071013

Cited by 23 publications

(34 citation statements)

References 33 publications

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“…Chemical modification and proteolysis studies of the apo-and holo-enzymes also indicated that a conformational change accompanies biotinylation [147]. The recent determination by NMR of the three-dimensional structure of the entire 1.3 S subunit of P. shermanii transcarboxylase, which functions as the carboxyl group carrier of this enzyme, also showed that the C-terminal half of this subunit is folded into a compact domain, consistent with the fold found both in the carboxyl carrier protein of E. coli ACC and in the lipoyl domains, to which this domain exhibits only 26-30 % sequence similarity [114]. Therefore it is predictable that the biotin carboxyl carrier domain of yeast PC would fold to a similar structure as the lipoyl domains [148], as there are remarkable sequence similarities between these two families of proteins [85].…”

Section: Structurementioning

confidence: 82%

Structure, function and regulation of pyruvate carboxylase

Jitrapakdee

Wallace

1999

Biochemical Journal

183

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Pyruvate carboxylase (PC; EC 6.4.1.1), a member of the biotin-dependent enzyme family, catalyses the ATP-dependent carboxylation of pyruvate to oxaloacetate. PC has been found in a wide variety of prokaryotes and eukaryotes. In mammals, PC plays a crucial role in gluconeogenesis and lipogenesis, in the biosynthesis of neurotransmitter substances, and in glucose-induced insulin secretion by pancreatic islets. The reaction catalysed by PC and the physical properties of the enzyme have been studied extensively. Although no high-resolution three-dimensional structure has yet been determined by X-ray crystallography, structural studies of PC have been conducted by electron microscopy, by limited proteolysis, and by cloning and sequencing of genes and cDNA encoding the enzyme. Most well characterized forms of active PC consist of four identical subunits arranged in a tetrahedron-like structure. Each subunit contains three functional domains: the biotin carboxylation domain, the transcarboxylation domain and the biotin carboxyl carrier domain. Different physiological conditions, including diabetes, hyperthyroidism, genetic obesity and postnatal development, increase the level of PC expression through transcriptional and translational mechanisms, whereas insulin inhibits PC expression. Glucocorticoids, glucagon and catecholamines cause an increase in PC activity or in the rate of pyruvate carboxylation in the short term. Molecular defects of PC in humans have recently been associated with four point mutations within the structural region of the PC gene, namely Val145 → Ala, Arg451 → Cys, Ala610 → Thr and Met743 → Thr.

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Section: Structurementioning

confidence: 82%

Structure, function and regulation of pyruvate carboxylase

Jitrapakdee

Wallace

1999

Biochemical Journal

183

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“…It was concluded that the protruding thumb between strands ␤2 and ␤3 is not critical for the interaction with BPL, but its presence is sufficient to prevent the E. coli biotinyl domain from becoming lipoylated (23). This effect may be limited to E. coli, because amino acids that constitute the thumb are not present in most other biotinyl domains (23,24). When we superimpose the biotinyl domain of acetyl-coenzyme A carboxylase from E. coli (25) (PDB code 1BDO) with the lipoyl domain of the modeled complex, the protruding thumb of the E. coli biotinyl domain clashes severely with Ta LplA (mostly the carboxyl-terminal side of ␣7) and, thus, binding of the biotinyl domain to LplA would be prevented.…”

Section: Resultsmentioning

confidence: 99%

Crystal Structure of Lipoate-Protein Ligase A Bound with the Activated Intermediate

Kim

Lee

et al. 2005

Journal of Biological Chemistry

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Lipoic acid is the covalently attached cofactor of several multicomponent enzyme complexes that catalyze key metabolic reactions. Attachment of lipoic acid to the lipoyl-dependent enzymes is catalyzed by lipoate-protein ligases (LPLs). In Escherichia coli, two distinct enzymes lipoate-protein ligase A (LplA) and lipB-encoded lipoyltransferase (LipB) catalyze independent pathways for lipoylation of the target proteins. The reaction catalyzed by LplA occurs in two steps. First, LplA activates exogenously supplied lipoic acid at the expense of ATP to lipoyl-AMP. Next, it transfers the enzymebound lipoyl-AMP to the ⑀-amino group of a specific lysine residue of the lipoyl domain to give an amide linkage. To gain insight into the mechanism of action by LplA, we have determined the crystal structure of Thermoplasma acidophilum LplA in three forms: (i) the apo form; (ii) the ATP complex; and (iii) the lipoyl-AMP complex. The overall fold of LplA bears some resemblance to that of the biotinyl protein ligase module of the E. coli biotin holoenzyme synthetase/bio repressor (BirA). Lipoyl-AMP is bound deeply in the bifurcated pocket of LplA and adopts a U-shaped conformation. Only the phosphate group and part of the ribose sugar of lipoyl-AMP are accessible from the bulk solvent through a tunnel-like passage, whereas the rest of the activated intermediate is completely buried inside the active site pocket. This first view of the activated intermediate bound to LplA allowed us to propose a model of the complexes between Ta LplA and lipoyl domains, thus shedding light on the target protein/lysine residue specificity of LplA.Lipoic acid (6,8-thioctic acid or 1,2-dithiolane-3-pentanoic acid), a covalently bound cofactor, is essential for function of several key enzymes involved in oxidative metabolism in most prokaryotic and eukaryotic organisms (1). The lipoylated proteins include pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, branched-chain 2-oxoacid dehydrogenase, and the glycine cleavage system (2). In the reaction catalyzed by the lipoylating enzymes, i.e. lipoate-protein ligases (LPLs), 3 the free carboxyl group of lipoic acid is attached via an amide linkage to the ⑀-amino group of a specific lysine residue of the lipoate-accepting protein domains (termed the lipoyl domains) of these multienzyme complexes (3). The lipoamide arm protruding from a tight ␤-turn of the structure of the lipoyl domains shuttles reaction intermediates among different active sites of the multienzyme complexes (2). Although the general role of lipoic acid as the covalently attached coenzyme has been known for decades, the mechanisms by which lipoic acid is synthesized and becomes linked to its cognate proteins continue to be elucidated. In Escherichia coli two independent LPL enzymes modify lipoyl domains (4). The best characterized lipoylating enzyme is E. coli lipoate-protein ligase A (LplA). LplA utilizes exogenously supplied free lipoic acid to modify the specific lysine of the lipoyl domain. In the first step, LplA catalyzes synthesis...

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“…Despite a wealth of sequence information available for biotinylated subunits from a range of organisms, as well as the knowledge of the three-dimensional structures of two members of this class of proteins~Athappilly & Hendrickson, 1995;Yao et al, 1997;Reddy et al, 1998!, Biotinylated refers to peptides that had been incubated with both an excess of the adenylate and the enzyme. Samples were prepared for MALDI-TOF as described in Materials and methods.…”

Section: Discussionmentioning

confidence: 99%

A minimal peptide substrate in biotin holoenzyme synthetase‐catalyzed biotinylation

1999

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The Escherichia coli biotin holoenzyme synthetase, BirA, catalyzes transfer of biotin to the epsilon amino group of a specific lysine residue of the biotin carboxyl carrier protein~BCCP! subunit of acetyl-CoA carboxylase. Sequences of naturally biotinylated substrates are highly conserved across evolutionary boundaries, and cross-species biotinylation has been demonstrated in several systems. To define the minimal substrate requirements in BirA-catalyzed biotinylation, we have measured the kinetics of modification of a 23-residue peptide previously identified by combinatorial methods. Although the sequence of the peptide bears little resemblance to the biotinylated sequence in BCCP, it is enzymatically biotinylated in vivo. Rates of biotin transfer to the 23-residue peptide are similar to those determined for BCCP. To further elucidate the sequence requirements for biotinylation, transient kinetic measurements were performed on a series of amino-and carboxy-terminal truncations of the 23-mer. The results, determined by stopped-flow fluorescence, allowed identification of a 14-residue peptide as the minimum required sequence. Additional support was obtained using matrix-assisted laser desorption ionization time-of-flight~MALDI-TOF! mass spectrometric analysis of peptides that had been incubated with an excess of biotinyl-59-adenylate intermediate and catalytic amounts of BirA. Results of these measurements indicate that while kinetically inactive truncations showed no significant shift in molecular mass to the values expected for biotinylated species, kinetically active truncations exhibited 100% biotinylation. The specificity constant~k cat 0K m ! governing BirA-catalyzed biotinylation of the 14-mer minimal substrate is similar to that determined for the natural substrate, BCCP. We conclude that the 14-mer peptide efficiently mimics the biotin acceptor function of the much larger protein domain normally recognized by BirA.

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Structural characterization of the entire 1.3S subunit of transcarboxylase from Propionibacterium shermanii

Cited by 23 publications

References 33 publications

Structure, function and regulation of pyruvate carboxylase

Structure, function and regulation of pyruvate carboxylase

Crystal Structure of Lipoate-Protein Ligase A Bound with the Activated Intermediate

A minimal peptide substrate in biotin holoenzyme synthetase‐catalyzed biotinylation

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