Protein engineering of pyruvate carboxylase

Sueda, Shinji; Islam, Md. Nurul; Kondo, Hiroki

doi:10.1111/j.1432-1033.2004.04051.x

Cited by 21 publications

(33 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…1B). This BC domain existed in a monomer-dimer equilibrium in solution, and was able to catalyze the hydrolysis of ATP in the presence of biotin, likely in its monomeric form (40). A chimera replacing the BC domain of G. thermodenitrificans PC with the BC subunit of A. aeolicus PC was also catalytically active (41).…”

Section: Resultsmentioning

confidence: 99%

Characterizing the Importance of the Biotin Carboxylase Domain Dimer for Staphylococcus aureus Pyruvate Carboxylase Catalysis

Chou²,

Choi

et al. 2013

Biochemistry

View full text Add to dashboard Cite

Biotin carboxylase (BC) is a conserved component among biotin-dependent carboxylases and catalyzes the MgATP-dependent carboxylation of biotin, using bicarbonate as the CO2 donor. Studies with E. coli BC have suggested long-range communication between the two active sites of a dimer, although its mechanism is not well understood. In addition, mutations in the dimer interface can produce stable monomers that are still catalytically active. A homologous dimer for the BC domain is observed in the structure of tetrameric pyruvate carboxylase (PC) holoenzyme. We have introduced site-specific mutations in the BC domain dimer interface of S. aureus PC (SaPC), equivalent to those used for E. coli BC, and also made chimeras replacing the SaPC BC domain with the E. coli BC subunit (EcBC chimera) or the yeast ACC BC domain (ScBC chimera). We assessed the catalytic activities of these mutants and characterized their oligomerization states by gel filtration and analytical ultracentrifugation experiments. The K442E mutant and the ScBC chimera disrupted the BC dimer and were catalytically inactive, while the F403A mutant and the EcBC chimera were still tetrameric and retained catalytic activity. The R54E mutant was also tetrameric but was catalytically inactive. Crystal structures of the R54E, F403A and K442E mutants showed that they were tetrameric in the crystal, with conformational changes near the mutation site as well as in the tetramer organization. We have also produced the isolated BC domain of SaPC. In contrast to E. coli BC, the SaPC BC domain is monomeric in solution and catalytically inactive.

show abstract

Section: Resultsmentioning

confidence: 99%

Characterizing the Importance of the Biotin Carboxylase Domain Dimer for Staphylococcus aureus Pyruvate Carboxylase Catalysis

Chou²,

Choi

et al. 2013

Biochemistry

View full text Add to dashboard Cite

show abstract

“…A BC domain deletion of Re PC forms dimers in the presence and absence of acetyl-CoA, confirming that the central domain of Re PC is insufficient to maintain tetramers (Figure 2). Truncation of the BC domain in Bacillus thermodenitrificans PC also results in the formation of active dimers (44). The same truncation in yeast pyruvate carboxylase I isoenzyme is unable to form tetramers and the truncated construct does not co-purify with wild-type tetramers upon co-expression (45).…”

Section: Discussionmentioning

confidence: 99%

Interaction between the Biotin Carboxyl Carrier Domain and the Biotin Carboxylase Domain in Pyruvate Carboxylase from Rhizobium etli

et al. 2011

View full text Add to dashboard Cite

Pyruvate carboxylase (PC) catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To effect catalysis, the tethered biotin of PC must gain access to active sites in both the biotin carboxylase domain and the carboxyl transferase domain. Previous studies have demonstrated that a mutation of threonine 882 to alanine in PC from Rhizobium etli renders the carboxyl transferase domain inactive and favors the positioning of biotin in the biotin carboxylase domain. We report the 2.4 Å resolution X-ray crystal structure of the Rhizobium etli PC T882A mutant which reveals the first high-resolution description of the domain interaction between the biotin carboxyl carrier protein domain and the biotin carboxylase domain. The overall quaternary arrangement of Rhizobium etli PC remains highly asymmetrical and is independent of the presence of allosteric activator. While biotin is observed in the biotin carboxylase domain, its access to the active site is precluded by the interaction between Arg353 and Glu248, revealing a mechanism for regulating carboxybiotin access to the BC domain active site. The binding location for the biotin carboxyl carrier protein domain demonstrates that tethered biotin cannot bind in the biotin carboxylase domain active site in the same orientation as free biotin, helping to explain the difference in catalysis observed between tethered biotin and free biotin substrates in biotin carboxylase enzymes. Electron density located in the biotin carboxylase domain active site is assigned to phosphonoacetate, offering a probable location for the putative carboxyphosphate intermediate formed during biotin carboxylation. The insights gained from the T882A Rhizobium etli PC crystal structure provide a new series of catalytic snapshots in PC and offer a revised perspective on catalysis in the biotin-dependent enzyme family.

show abstract

“…Thus, it is possible that, within the cell, RNIs can oxidize cysteine thiols to generate mixed intermolecular disulfide bonds (49). Such mixed disulfide formation may interfere with the proper assembly and function of oligomeric protein complexes, such as AccA3 and Pca (46,47), or alter the stability of proteins such as KatG, in which isosteric mutation of a highly conserved cysteine to serine resulted in increased proteolytic degradation (50).…”

Section: Resultsmentioning

confidence: 99%

“…The largest biotinylated species contained both AccA3 and catalase (encoded by katG), a protein involved in antioxidant defense, also identified as a target for S-nitrosylation. AccA3 and Pca are structurally homologous, biotin-dependent, multidomain complexes that contain highly conserved cysteine residues, and whose assembly is associated with the self-interaction of two biotin-containing domains (encoded by accA3 and pca, respectively) (46,47). In solution, KatG similarly exists as a homodimer and contains a highly conserved cysteine (48).…”

Section: Resultsmentioning

confidence: 99%

S-nitroso proteome ofMycobacterium tuberculosis: Enzymes of intermediary metabolism and antioxidant defense

Rhee

Erdjument‐Bromage

Tempst

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

158

139

View full text Add to dashboard Cite

The immune response to Mycobacterium tuberculosis (Mtb) includes expression of nitric oxide (NO) synthase (NOS)2, whose products can kill Mtb in vitro with a molar potency greater than that of many conventional antitubercular agents. However, the targets of reactive nitrogen intermediates (RNIs) in Mtb are unknown. One major action of RNIs is protein S-nitrosylation. Here, we describe, to our knowledge, the first proteomic analysis of S-nitrosylation in a whole organism after treating Mtb with bactericidal concentrations of RNIs. The 29 S-nitroso proteins identified are all enzymes, mostly serving intermediary metabolism, lipid metabolism, and͞or antioxidant defense. Many are essential or implicated in virulence, including defense against RNIs. For each of two target enzymes tested, lipoamide dehydrogenase and mycobacterial proteasome ATPase, S-nitrosylation caused enzyme inhibition. Moreover, endogenously biotinylated proteins were driven into mixed disulfide complexes. Targeting of metabolic enzymes and antioxidant defenses by means of protein S-nitrosylation and mixed disulfide bonding may contribute to the antimycobacterial actions of RNIs.nitric oxide ͉ biotin ͉ lipoamide dehydrogenase ͉ mycobacterial proteasome ATPase

show abstract

Protein engineering of pyruvate carboxylase

Cited by 21 publications

References 38 publications

Characterizing the Importance of the Biotin Carboxylase Domain Dimer for Staphylococcus aureus Pyruvate Carboxylase Catalysis

Characterizing the Importance of the Biotin Carboxylase Domain Dimer for Staphylococcus aureus Pyruvate Carboxylase Catalysis

Interaction between the Biotin Carboxyl Carrier Domain and the Biotin Carboxylase Domain in Pyruvate Carboxylase from Rhizobium etli

S-nitroso proteome ofMycobacterium tuberculosis: Enzymes of intermediary metabolism and antioxidant defense

Contact Info

Product

Resources

About