Article

Crystal structure of Escherichia coli crotonobetainyl-CoA: carnitine CoA-transferase (CaiB) and its complexes with CoA and carnitinyl-CoA.

Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Biochemistry (Impact Factor: 3.38). 05/2005; 44(15):5728-38. DOI: 10.1021/bi047656f
Source: PubMed

ABSTRACT L-Carnitine (R-[-]-3-hydroxy-4-trimethylaminobutyrate) is found in both eukaryotic and prokaryotic cells and participates in diverse processes including long-chain fatty-acid transport and osmoprotection. The enzyme crotonobetainyl/gamma-butyrobetainyl-CoA:carnitine CoA-transferase (CaiB; E.C. 2.8.3.-) catalyzes the first step in carnitine metabolism, leading to the final product gamma-butyrobetaine. The crystal structures of Escherichia coli apo-CaiB, as well as its Asp169Ala mutant bound to CoA and to carnitinyl-CoA, have been determined and refined to 1.6, 2.4, and 2.4 A resolution, respectively. CaiB is composed of two identical circular chains that together form an intertwined dimer. Each monomer consists of a large domain, containing a Rossmann fold, and a small domain. The monomer and dimer resemble those of formyl-CoA transferase from Oxalobacter formigenes, as well as E. coli YfdW, a putative type-III CoA transferase of unknown function. The CoA cofactor-binding site is formed at the interface of the large domain of one monomer and the small domain from the second monomer. Most of the protein-CoA interactions are formed with the Rossmann fold domain. While the location of cofactor binding is similar in the three proteins, the specific CoA-protein interactions vary somewhat between CaiB, formyl-CoA transferase, and YfdW. CoA binding results in a change in the relative positions of the large and small domains compared with apo-CaiB. The observed carnitinyl-CoA product in crystals of the CaiB Asp169Ala mutant cocrystallized with crotonoyl-CoA and carnitine could result from (i) a catalytic mechanism involving a ternary enzyme-substrate complex, independent of a covalent anhydride intermediate with Asp169, (ii) a spontaneous reaction of the substrates in solution, followed by binding to the enzyme, or (iii) an involvement of another residue substituting functionally for Asp169, such as Glu23.

0 Bookmarks
 · 
64 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: The act gene of Variovorax paradoxus TBEA6 encodes a succinyl-CoA:3-sulfinopropionate CoA-transferase (2.8.3.x), which catalyzes the activation of 3-sulfinopropionate (3SP), an intermediate during 3,3'-thiodipropionate (TDP) degradation. In a previous study, accumulation of 3SP was observed in a Tn5::mob-induced mutant defective in growth on TDP. In contrast to the wild type and all other obtained mutants, this mutant showed no growth when 3SP was applied as a sole source of carbon and energy. The transposon Tn5::mob inserted in a gene showing high homology to class III CoA-transferases. In the current study, analyses of the translation product clearly allocated ActTBEA6 to this protein family. The predicted secondary structure indicates the lack of a C-terminal α-helix. ActTBEA6 was heterologously expressed in E. coli Lemo21 (DE3) and was then purified applying Ni-NTA affinity chromatography. Analytical size-exclusion chromatography revealed a homodimeric structure with a molecular mass of 96 kDa ± 3 kDa. Enzyme assays identified succinyl-CoA, itaconyl-CoA and glutaryl-CoA as potential CoA donors and unequivocally verified the conversion of 3SP to 3SP-CoA. Kinetic studies revealed an apparent Vmax of 44.6 μmol min(-1) mg(-1) for succinyl-CoA which corresponds to a turnover number of 36.0 s(-1) per subunit of ActTBEA6. For 3SP, the apparent Vmax was determined as 46.8 μmol min(-1) mg(-1) which corresponds to a turnover number of 37.7 s(-1) per subunit of ActTBEA6. The apparent Km values were 0.08 mM for succinyl-CoA and 5.9 mM for 3SP. Nonetheless, mutant V. paradoxus Δact did not reproduce the phenotype of the Tn5::mob induced mutant. This defined deletion mutant was able to utilize TDP or 3SP as sole carbon source like the wild type. Complementation of the Tn5::mob induced mutant with pBBR1MCS5::acdDPN7 partially restored growth on 3SP, which indicated a polar effect of the Tn5::mob transposon on acdTBEA6, located downstream of actTBEA6.
    Journal of bacteriology 06/2013; · 3.94 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Many food plants accumulate oxalate, which humans absorb but do not metabolize, leading to the formation of urinary stones. The commensal bacterium Oxalobacter formigenes consumes oxalate by converting it to oxalyl-CoA, which is decarboxylated by oxalyl-CoA decarboxylase (OXC). OXC and the class III CoA-transferase formyl-CoA:oxalate CoA-transferase (FCOCT) are widespread among bacteria, including many that have no apparent ability to degrade or to resist external oxalate. The EvgA acid response regulator activates transcription of the Escherichia coli yfdXWUVE operon encoding YfdW (FCOCT), YfdU (OXC), and YfdE, a class III CoA-transferase that is [Formula: see text]30% identical to YfdW. YfdW and YfdU are necessary and sufficient for oxalate-induced protection against a subsequent acid challenge; neither of the other genes has a known function. We report the purification, in vitro characterization, 2.1-Å crystal structure, and functional assignment of YfdE. YfdE and UctC, an orthologue from the obligate aerobe Acetobacter aceti, perform the reversible conversion of acetyl-CoA and oxalate to oxalyl-CoA and acetate. The annotation of YfdE as acetyl-CoA:oxalate CoA-transferase (ACOCT) expands the scope of metabolic pathways linked to oxalate catabolism and the oxalate-induced acid tolerance response. FCOCT and ACOCT active sites contain distinctive, conserved active site loops (the glycine-rich loop and the GNxH loop, respectively) that appear to encode substrate specificity.
    PLoS ONE 01/2013; 8(7):e67901. · 3.53 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The human bile acid pool composition is composed of both primary bile acids (cholic acid and chenodeoxycholic acid) and secondary bile acids (deoxycholic acid and lithocholic acid). Secondary bile acids are formed by the 7α-dehydroxylation of primary bile acids carried out by intestinal anaerobic bacteria. We have previously described a multistep biochemical pathway in Clostridium scindens that is responsible for bile acid 7α-dehydroxylation. We have identified a large (12 kb) bile acid inducible (bai) operon in this bacterium that encodes eight genes involved in bile acid 7α-dehydroxylation. However, the function of the baiF gene product in this operon has not been elucidated. In the current study, we cloned and expressed the baiF gene in E. coli and discovered it has bile acid CoA transferase activity. In addition, we discovered a second bai operon encoding three genes. The baiK gene in this operon was expressed in E. coli and found to encode a second bile acid CoA transferase. Both bile acid CoA transferases were determined to be members of the type III family by amino acid sequence comparisons. Both bile acid CoA transferases had broad substrate specificity, except the baiK gene product, which failed to use lithocholyl-CoA as a CoA donor. Primary bile acids are ligated to CoA via an ATP-dependent mechanism during the initial steps of 7α-dehydroxylation. The bile acid CoA transferases conserve the thioester bond energy, saving the cell ATP molecules during bile acid 7α-dehydroxylation. ATP-dependent CoA ligation is likely quickly supplanted by ATP-independent CoA transfer.
    The Journal of Lipid Research 01/2012; 53(1):66-76. · 4.39 Impact Factor