Molecular biology of biotin attachment to proteins.
Enzymatic attachment of biotin to proteins requires the interaction of a distinct domain of the acceptor protein (the "biotin domain") with the enzyme, biotin protein ligase, that catalyzes this essential and rare post-translational modification. Both biotin domains and biotin protein ligases are very strongly conserved throughout biology. This review concerns the protein structures and mechanisms involved in the covalent attachment of biotin to proteins. (+info
A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation.
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-5'-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)/Km) 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. (+info
Light-dependent changes in redox status of the plastidic acetyl-CoA carboxylase and its regulatory component.
Plastidic acetyl-CoA carboxylase (ACCase; EC 126.96.36.199), which catalyses the synthesis of malonyl-CoA and is the regulatory enzyme of fatty acid synthesis, is activated by light, presumably under redox regulation. To obtain evidence of redox regulation in vivo, the activity of ACCase was examined in pea chloroplasts isolated from plants kept in darkness (dark-ACCase) or after exposure to light for 1 h (light-ACCase) in the presence or absence of a thiol-reducing agent, dithiothreitol (DTT). The protein level was similar for light-ACCase and dark-ACCase, but the activity of light-ACCase in the absence of DTT was approx. 3-fold that of dark-ACCase. The light-ACCase and dark-ACCase were activated approx. 2-fold and 6-fold by DTT respectively, indicating that light-ACCase was in a much more reduced, active form than the dark-ACCase. This is the first demonstration of the light-dependent reduction of ACCase in vivo. Measurement of the activities of ACCase, carboxyltransferase and biotin carboxylase in the presence and absence of DTT, and the thiol-oxidizing agent, 5, 5'-dithiobis-(2-nitrobenzoic) acid, revealed that the carboxyltransferase reaction, but not the biotin carboxylase reaction, was redox-regulated. The cysteine residue(s) responsible for redox regulation probably reside on the carboxyltransferase component. Measurement of the pH dependence of biotin carboxylase and carboxyltransferase activities in the ACCase suggested that both components affect the activity of ACCase in vivo at a physiological pH range. These results suggest that the activation of ACCase by light is caused partly by the pH-dependent activation of two components and by the reductive activation of carboxyltransferase. (+info
Rickettsia prowazekii transports UMP and GMP, but not CMP, as building blocks for RNA synthesis.
Rickettsia prowazekii, the etiological agent of epidemic typhus, is an obligate intracellular bacterium and is apparently unable to synthesize ribonucleotides de novo. Here, we show that as an alternative, isolated, purified R. prowazekii organisms transported exogenous uridyl- and guanylribonucleotides and incorporated these labeled precursors into their RNA in a rifampin-sensitive manner. Transport systems for nucleotides, which we have shown previously and show here are present in rickettsiae, have never been reported in free-living bacteria, and the usual nucleobase and nucleoside transport systems are absent in rickettsiae. There was a clear preference for the monophosphate form of ribonucleotides as the transported substrate. In contrast, rickettsiae did not transport cytidylribonucleotides. The source of rickettsial CTP appears to be the transport of UMP followed by its phosphorylation and the amination of intrarickettsial UTP to CTP by CTP synthetase. A complete schema of nucleotide metabolism in rickettsiae is presented that is based on a combination of biochemical, physiological, and genetic information. (+info
X-ray crystal structure of aminoimidazole ribonucleotide synthetase (PurM), from the Escherichia coli purine biosynthetic pathway at 2.5 A resolution.
BACKGROUND: The purine biosynthetic pathway in procaryotes enlists eleven enzymes, six of which use ATP. Enzymes 5 and 6 of this pathway, formylglycinamide ribonucleotide (FGAR) amidotransferase (PurL) and aminoimidazole ribonucleotide (AIR) synthetase (PurM) utilize ATP to activate the oxygen of an amide within their substrate toward nucleophilic attack by a nitrogen. AIR synthetase uses the product of PurL, formylglycinamidine ribonucleotide (FGAM) and ATP to make AIR, ADP and P(i). RESULTS: The structure of a hexahistidine-tagged PurM has been solved by multiwavelength anomalous diffraction phasing techniques using protein containing 28 selenomethionines per asymmetric unit. The final model of PurM consists of two crystallographically independent dimers and four sulfates. The overall R factor at 2.5 A resolution is 19.2%, with an R(free) of 26.4%. The active site, identified in part by conserved residues, is proposed to be a long groove generated by the interaction of two monomers. A search of the sequence databases suggests that the ATP-binding sites between PurM and PurL may be structurally conserved. CONCLUSIONS: The first structure of a new class of ATP-binding enzyme, PurM, has been solved and a model for the active site has been proposed. The structure is unprecedented, with an extensive and unusual sheet-mediated intersubunit interaction defining the active-site grooves. Sequence searches suggest that two successive enzymes in the purine biosynthetic pathway, proposed to use similar chemistries, will have similar ATP-binding domains. (+info
The biotin domain peptide from the biotin carboxyl carrier protein of Escherichia coli acetyl-CoA carboxylase causes a marked increase in the catalytic efficiency of biotin carboxylase and carboxyltransferase relative to free biotin.
Acetyl-CoA carboxylase catalyzes the first committed step in the biosynthesis of long-chain fatty acids. The Escherichia coli form of the enzyme consists of a biotin carboxylase activity, a biotin carboxyl carrier protein, and a carboxyltransferase activity. The C-terminal 87 amino acids of the biotin carboxyl carrier protein (BCCP87) form a domain that can be independently expressed, biotinylated, and purified (Chapman-Smith, A., Turner, D. L., Cronan, J. E., Morris, T. W., and Wallace, J. C. (1994) Biochem. J. 302, 881-887). The ability of the biotinylated form of this 87-residue protein (holoBCCP87) to act as a substrate for biotin carboxylase and carboxyltransferase was assessed and compared with the results with free biotin. In the case of biotin carboxylase holoBCCP87 was an excellent substrate with a K(m) of 0.16 +/- 0.05 mM and V(max) of 1000.8 +/- 182.0 min(-1). The V/K or catalytic efficiency of biotin carboxylase with holoBCCP87 as substrate was 8000-fold greater than with biotin as substrate. Stimulation of the ATP synthesis reaction of biotin carboxylase where carbamyl phosphate reacted with ADP by holoBCCP87 was 5-fold greater than with an equivalent amount of biotin. The interaction of holoBCCP87 with carboxyltransferase was characterized in the reverse direction where malonyl-CoA reacted with holoBCCP87 to form acetyl-CoA and carboxyholoBCCP87. The K(m) for holoBCCP87 was 0.45 +/- 0.07 mM while the V(max) was 2031.8 +/- 231.0 min(-1). The V/K or catalytic efficiency of carboxyltransferase with holoBCCP87 as substrate is 2000-fold greater than with biotin as substrate. (+info
Biotin protein ligase from Saccharomyces cerevisiae. The N-terminal domain is required for complete activity.
Catalytically active biotin protein ligase from Saccharomyces cerevisiae (EC 188.8.131.52) was overexpressed in Escherichia coli and purified to near homogeneity in three steps. Kinetic analysis demonstrated that the substrates ATP, biotin, and the biotin-accepting protein bind in an ordered manner in the reaction mechanism. Treatment with any of three proteases of differing specificity in vitro revealed that the sequence between residues 240 and 260 was extremely sensitive to proteolysis, suggesting that it forms an exposed linker between an N-terminal 27-kDa domain and the C-terminal 50-kDa domain containing the active site. The protease susceptibility of this linker region was considerably reduced in the presence of ATP and biotin. A second protease-sensitive sequence, located in the presumptive catalytic site, was protected against digestion by the substrates. Expression of N-terminally truncated variants of the yeast enzyme failed to complement E. coli strains defective in biotin protein ligase activity. In vitro assays performed with purified N-terminally truncated enzyme revealed that removal of the N-terminal domain reduced BPL activity by greater than 3500-fold. Our data indicate that both the N-terminal domain and the C-terminal domain containing the active site are necessary for complete catalytic function. (+info
Using genomic information to investigate the function of ThiI, an enzyme shared between thiamin and 4-thiouridine biosynthesis.
The gene thiI encodes a protein (ThiI) that plays a role in the transfer of sulfur from cysteine to both thiamin and 4-thiouridine, but the reaction catalyzed by ThiI remains undetermined. Based upon sequence alignments, ThiI shares a unique "P-loop" motif with the PPi synthetase family, four enzymes that catalyze adenylation and subsequent substitution of carbonyl oxygens. To test whether or not this motif is critical for ThiI function, the Asp in the motif was converted to Ala (D189A), and a screen for in vivo 4-thiouridine production revealed the altered enzyme to be inactive. Further scrutiny of sequence data and the crystal structures of two members of the PPi synthetase family implicated Lys321 in the proposed adenylation function of ThiI, and the critical nature of Lys321 has been demonstrated by site-directed mutagenesis and genetic screening. Our results, then, indicate that ThiI catalyzes the adenylation of a substrate at the expense of ATP, a narrowing of possible reactions that provides a strong new basis for deducing the early steps in the transfer of sulfur from cysteine to both thiamin and 4-thiouridine. (+info