Computer-assisted Docking of Flavodoxin with the ATP:Co(I)rrinoid Adenosyltransferase (CobA) Enzyme Reveals Residues Critical for Protein-Protein Interactions but Not for Catalysis

The activity of the housekeeping ATP:co(I)rrinoid adenosyltransferase (CobA) enzyme of Salmonella enterica sv. Typhimurium is required to adenosylate de novo biosynthetic intermediates of adenosylcobalamin and to salvage incomplete and complete corrinoids from the environment of this bacterium. In vitro, reduced flavodoxin (FldA) provides an electron to generate the co(I)rrinoid substrate in the CobA active site. To understand how CobAand FldA interact, a computer model of aCobA-FldA complex was generated. This model was used to guide the introduction of mutations into CobA using site-directed mutagenesis and the synthesis of a peptide mimic of FldA. Residues Arg-9 and Arg-165 of CobA were critical for FldA-dependent adenosylation but were catalytically as competent as the wild-type protein when cob(I)alamin was provided as substrate. These results indicate that Arg-9 and Arg-165 are important for CobA_FldA docking but not to catalysis. A truncation of the 9-amino acid N-terminal helix of CobA reduced its FldA-dependent cobalamin adenosyltransferase activity by 97.4%. The same protein, however, had a 4-fold higher specific activity than the native enzyme when cob(I)alamin was generated chemically in situ

Nε−Lysine Acetylation of a Bacterial Transcription Factor Inhibits Its DNA-Binding Activity

Evidence suggesting that eukaryotes and archaea use reversible Nε-lysine (Nε-Lys) acetylation to modulate gene expression has been reported, but evidence for bacterial use of Nε-Lys acetylation for this purpose is lacking. Here, we report data in support of the notion that bacteria can control gene expression by modulating the acetylation state of transcription factors (TFs). We screened the E. coli proteome for substrates of the bacterial Gcn5-like protein acetyltransferase (Pat). Pat acetylated four TFs, including the RcsB global regulatory protein, which controls cell division, and capsule and flagellum biosynthesis in many bacteria. Pat acetylated residue Lys180 of RcsB, and the NAD+-dependent Sir2 (sirtuin)-like protein deacetylase (CobB) deacetylated acetylated RcsB (RcsBAc), demonstrating that Nε-Lys acetylation of RcsB is reversible. Analysis of RcsBAc and variant RcsB proteins carrying substitutions at Lys180 provided biochemical and physiological evidence implicating Lys180 as a critical residue for RcsB DNA-binding activity. These findings further the likelihood that reversible Nε-Lys acetylation of transcription factors is a mode of regulation of gene expression used by all cells

Purification and Initial Biochemical Characterization of ATP:Cob(I)alamin Adenosyltransferase (EutT) Enzyme of \u3ci\u3eSalmonella enterica\u3c/i\u3e

ATP:cob(I)alamin adenosyltransferase (EutT) of Salmonella enterica was overproduced and enriched to ~70% homogeneity, and its basic kinetic parameters were determined. Abundant amounts of EutT protein were produced, but all of it remained insoluble. Soluble active EutT protein (~70% homogeneous) was obtained after treatment with detergent. Under conditions in which cobalamin (Cbl) was saturating, Km(ATP) = 10 µM, kcat = 0.03 s-1, and Vmax = 54.5 nM min-1. Similarly, under conditions in which MgATPwas saturating,Km(Cbl) = 4.1µM, kcat = 0.06 s-1, andVmax= 105 nM min-1. Unlike other ATP:co(I)rrinoid adenosyltransferases in the cell (i.e. CobA and PduO), EutT activity was \u3e50-fold higher with ATP versus GTP, and EutT retained 80% of its activity with ADP substituted for ATP and was completely inactive with AMP as substrate, indicating that the enzyme requires the β-phosphate group of the nucleotide substrate. The data suggest that the amino group of adenine might play a role in nucleotide recognition and/or binding. Unlike the housekeeping CobA enzyme, EutT was not inhibited by inorganic tripolyphosphate (PPPi). Results from 31P NMR spectroscopy studies identified PPi and Pi as by-products of the EutT reaction. In the absence of Cbl, EutT cleaved ATP into adenosine and PPPi, suggesting that PPPi is broken down into PPi and Pi. Electron transfer protein partners for EutT were not encoded by the eut operon. EutT-dependent activity was detected in cell-free extracts of cobA strains enriched for EutT when FMN and NADH were used to reduce cob(III)alamin to cob(I)alamin

Purification and Initial Biochemical Characterization of ATP:Cob(I)alamin Adenosyltransferase (EutT) Enzyme of \u3ci\u3eSalmonella enterica\u3c/i\u3e

ATP:cob(I)alamin adenosyltransferase (EutT) of Salmonella enterica was overproduced and enriched to ~70% homogeneity, and its basic kinetic parameters were determined. Abundant amounts of EutT protein were produced, but all of it remained insoluble. Soluble active EutT protein (~70% homogeneous) was obtained after treatment with detergent. Under conditions in which cobalamin (Cbl) was saturating, Km(ATP) = 10 μM, kcat = 0.03 s–1, and Vmax = 54.5 nM min–1. Similarly, under conditions in which MgATPwas saturating,Km(Cbl)= 4.1μM, kcat = 0.06 s–1, andVmax= 105 nM min–1. Unlike other ATP:co(I)rrinoid adenosyltransferases in the cell (i.e. CobA and PduO), EutT activity was \u3e50-fold higher with ATP versus GTP, and EutT retained 80% of its activity with ADP substituted for ATP and was completely inactive with AMP as substrate, indicating that the enzyme requires the β-phosphate group of the nucleotide substrate. The data suggest that the amino group of adenine might play a role in nucleotide recognition and/or binding. Unlike the housekeeping CobA enzyme, EutT was not inhibited by inorganic tripolyphosphate (PPPi). Results from 31P NMR spectroscopy studies identified PPi and Pi as by-products of the EutT reaction. In the absence of Cbl, EutT cleaved ATP into adenosine and PPPi, suggesting that PPPi is broken down into PPi and Pi. Electron transfer protein partners for EutT were not encoded by the eut operon. EutT-dependent activity was detected in cell-free extracts of cobA strains enriched for EutT when FMN and NADH were used to reduce cob(III)alamin to cob(I)alamin

In vitro analysis of the interactions between the PocR regulatory protein and the promoter region of the cobalamin biosynthetic (cob) operon of Salmonella typhimurium LT2.

The PocR protein of Salmonella typhimurium LT2 was overexpressed and used to demonstrate in vitro that it specifically binds to the cobalamin biosynthetic operon (cob) promoter region. Evidence is presented to show that PocR DNA-binding activity in vitro is regulated by the effector molecule 1,2-propanediol. Deletion analysis of the cob promoter (Pcob) suggested that two regions upstream of the promoter are needed for optimal activation of Pcob by PocR in vivo. DNase I footprinting experiments demonstrated that PocR binds to two sites within Pcob. The transcription initiation site of cob mRNA in response to 1,2-propanediol was identified and shown to be different from the one reported for transcription initiation under anoxic conditions in the absence of 1,2-propanediol

The eutD Gene of Salmonella enterica Encodes a Protein with Phosphotransacetylase Enzyme Activity

The EutD protein of Salmonella enterica is homologous to the catalytic domain of the phosphotransacetylase (Pta) enzyme. The Pta-like activity level of the EutD enzyme compared favorably to that of other Pta enzymes. High-pressure liquid chromatography and mass spectrometry verified that acetyl-coenzyme A was the product of the reaction. The EutD protein restored growth of an S. enterica pta strain on acetate as the source of carbon and energy