Timothy J. Larson

Purification and characterization of glpX-encoded fructose 1, 6-bisphosphatase, a new enzyme of the glycerol 3-phosphate regulon of Escherichia coli.

In Escherichia coli, gene products of the glp regulon mediate utilization of glycerol and sn-glycerol 3-phosphate. The glpFKX operon encodes glycerol diffusion facilitator, glycerol kinase, and as shown here, a fructose 1,6-bisphosphatase that is distinct from the previously described fbp-encoded enzyme. The purified enzyme was dimeric, dependent on Mn(2+) for activity, and exhibited an apparent K(m) of 35 microM for fructose 1,6-bisphosphate. The enzyme was inhibited by ADP and phosphate and activated by phosphoenolpyruvate.

Characterization of two genes, glpQ and ugpQ, encoding glycerophosphoryl diester phosphodiesterases of Escherichia coli.

SummaryThe nucleotide sequences of the glpQ and ugpQ genes of Escherichia coli, which both encode glycerophosphoryl diester phosphodiesterases, were determined. The glpQ gene encodes a periplasmic enzyme of 333 amino acids, produced initially with a 25 residue long signal sequence, while ugpQ codes for a cytoplasmic protein of 247 amino acids. Despite differences in size and cellular location, significant similarity in the primary structures of the two enzymes was found suggesting a common evolutionary origin. The 3′ end of the ugpQ gene overlaps an open reading frame that is transcribed in the opposite direction. This open reading frame encodes a polypeptide with an unusual composition, i.e., 46 of the 146 amino acids are Gln or Asn. This polypeptide and the UgpQ protein were identified in an in vitro transcription/translation system as proteins with apparent molecular weights of 19.5 and 27 kDa, respectively.

Functional Diversity of the Rhodanese Homology Domain THE ESCHERICHIA COLI ybbB GENE ENCODES A SELENOPHOSPHATE-DEPENDENT tRNA 2-SELENOURIDINE SYNTHASE

Escherichia coli has eight genes predicted to encode sulfurtransferases having the active site consensus sequence Cys-Xaa-Xaa-Gly. One of these genes, ybbB, is frequently found within bacterial operons that contain selD, the selenophosphate synthetase gene, suggesting a role in selenium metabolism. We show that ybbB is required in vivo for the specific substitution of selenium for sulfur in 2-thiouridine residues in E. coli tRNA. This modified tRNA nucleoside, 5-methylaminomethyl-2-selenouridine (mnm5se2U), is located at the wobble position of the anticodons of tRNALys, tRNAGlu, and tRNA1Gln. Nucleoside analysis of tRNAs from wild-type and ybbB mutant strains revealed that production of mnm5se2U is lost in the ybbB mutant but that 5-methylaminomethyl-2-thiouridine, the mnm5se2U precursor, is unaffected by deletion of ybbB. Thus, ybbB is not required for the initial sulfurtransferase reaction but rather encodes a 2-selenouridine synthase that replaces a sulfur atom in 2-thiouridine in tRNA with selenium. Purified 2-selenouridine synthase containing a C-terminal His6 tag exhibited spectral properties consistent with tRNA bound to the enzyme. In vitro mnm5se2U synthesis is shown to be dependent on 2-selenouridine synthase, SePO3, and tRNA. Finally, we demonstrate that the conserved Cys97 (but not Cys96) in the rhodanese sequence motif Cys96-Cys97-Xaa-Xaa-Gly is required for 2-selenouridine synthase in vivo activity. These data are consistent with the ybbB gene encoding a tRNA 2-selenouridine synthase and identifies a new role for the rhodanese homology domain in enzymes.

Escherichia coli GlpE Is a Prototype Sulfurtransferase for the Single-Domain Rhodanese Homology Superfamily

BACKGROUND Rhodanese domains are structural modules occurring in the three major evolutionary phyla. They are found as single-domain proteins, as tandemly repeated modules in which the C-terminal domain only bears the properly structured active site, or as members of multidomain proteins. Although in vitro assays show sulfurtransferase or phosphatase activity associated with rhodanese or rhodanese-like domains, specific biological roles for most members of this homology superfamily have not been established. RESULTS Eight ORFs coding for proteins consisting of (or containing) a rhodanese domain bearing the potentially catalytic Cys have been identified in the Escherichia coli K-12 genome. One of these codes for the 12-kDa protein GlpE, a member of the sn-glycerol 3-phosphate (glp) regulon. The crystal structure of GlpE, reported here at 1.06 A resolution, displays alpha/beta topology based on five beta strands and five alpha helices. The GlpE catalytic Cys residue is persulfurated and enclosed in a structurally conserved 5-residue loop in a region of positive electrostatic field. CONCLUSIONS Relative to the two-domain rhodanese enzymes of known three-dimensional structure, GlpE displays substantial shortening of loops connecting alpha helices and beta sheets, resulting in radical conformational changes surrounding the active site. As a consequence, GlpE is structurally more similar to Cdc25 phosphatases than to bovine or Azotobacter vinelandii rhodaneses. Sequence searches through completed genomes indicate that GlpE can be considered to be the prototype structure for the ubiquitous single-domain rhodanese module.

Cloning and nucleotide sequence of the fabD gene encoding malonyl coenzyme A‐acyl carrier protein transacylase of Escherichia coli

We report the cloning and nucleotide sequence of the gene encoding malonyl coenzyme A‐acyl carrier protein transacylase of Escherichia coli, Malonyl transacylase has been overexpressed 155‐fold compared to a wild‐type strain, Overexpression of this enzyme alters the fatty acid composition of a wild‐type E. coli strain; increased amounts of cis‐vaccenate are incorporated into the membrane phospholipids.

A new vector-host system for construction of lacZ transcriptional fusions where only low-level gene expression is desirable ☆

We improved a multicopy vector, pRS415 [Simons et al., Gene 53 (1987) 85-96], for use in operon fusion constructions by introducing a new multiple cloning site (MCS) containing eight unique restriction sites upstream from the promoterless reporter gene lacZ. In order to reduce plasmid copy number, a new Escherichia coli strain SP2 (pcnB, delta lac, recA) was constructed. This strain permits analysis of fusions in cases where high gene dosage may be detrimental.

Application of AgaR repressor and dominant repressor variants for verification of a gene cluster involved in N‐acetylgalactosamine metabolism in Escherichia coli K‐12

The agaZVWEFASYBCDI gene cluster encodes the phosphotransferase systems and enzymes responsible for the uptake and metabolism of N‐acetylgalactosamine and galactosamine in Escherichia coli. In some strains of E. coli, particularly the common K‐12 strain, a portion of this cluster is missing because of a site‐specific recombination event that occurred between sites in agaW and agaA. Strains that have undergone this recombination event have lost the ability to utilize either N‐acetylgalactosamine or galactosamine as sole sources of carbon. Divergently transcribed from this gene cluster is the gene agaR encoding a transcriptional repressor belonging to the DeoR/GlpR family of transcriptional regulators. Promoters upstream of agaR, agaZ and agaS were characterized. All three promoters had elevated activity in the presence of N‐acetylgalactosamine or galactosamine, were regulated in vivo by AgaR and possessed specific DNA‐binding sites for AgaR upstream from the start sites of transcription as determined by DNase I footprinting. In vivo analysis and DNase I footprinting indicated that the promoter specific for agaZ also requires activation by cAMP‐CRP. Previous work with GlpR and other members of the DeoR/GlpR family have identified highly conserved amino acid residues that function in DNA‐binding or response to inducer. These residues of AgaR were targeted for site‐directed mutagenesis and yielded variants of AgaR that were either negatively dominant or non‐inducible. The apparent ability to produce negatively dominant and non‐inducible variants of proteins of the DeoR/GlpR family of currently unknown function will likely facilitate screening for function.

The identification of a novel protein involved in molybdenum cofactor biosynthesis in Escherichia coli.

Background: In Moco biosynthesis, sulfur is transferred from l-cysteine to MPT synthase, catalyzing the conversion of cPMP to MPT. Results: The rhodanese-like protein YnjE is a novel protein involved in Moco biosynthesis. Conclusion: YnjE enhances the rate of conversion of cPMP to MPT and interacts with MoeB and IscS. Significance: To understand the mechanism of sulfur transfer and the role of rhodaneses in the cell. In the second step of the molybdenum cofactor (Moco) biosynthesis in Escherichia coli, the l-cysteine desulfurase IscS was identified as the primary sulfur donor for the formation of the thiocarboxylate on the small subunit (MoaD) of MPT synthase, which catalyzes the conversion of cyclic pyranopterin monophosphate to molybdopterin (MPT). Although in Moco biosynthesis in humans, the thiocarboxylation of the corresponding MoaD homolog involves two sulfurtransferases, an l-cysteine desulfurase, and a rhodanese-like protein, the rhodanese-like protein in E. coli remained enigmatic so far. Using a reverse approach, we identified a so far unknown sulfurtransferase for the MoeB-MoaD complex by protein-protein interactions. We show that YnjE, a three-domain rhodanese-like protein from E. coli, interacts with MoeB possibly for sulfur transfer to MoaD. The E. coli IscS protein was shown to specifically interact with YnjE for the formation of the persulfide group on YnjE. In a defined in vitro system consisting of MPT synthase, MoeB, Mg-ATP, IscS, and l-cysteine, YnjE was shown to enhance the rate of the conversion of added cyclic pyranopterin monophosphate to MPT. However, YnjE was not an enhancer of the cysteine desulfurase activity of IscS. This is the first report identifying the rhodanese-like protein YnjE as being involved in Moco biosynthesis in E. coli. We believe that the role of YnjE is to make the sulfur transfer from IscS for Moco biosynthesis more specific because IscS is involved in a variety of different sulfur transfer reactions in the cell.

Multiple promoters are responsible for transcription of the glpEGR operon of Escherichia coli K-12

The transcriptional organization of the glpEGR genes of Escherichia coli was studied. Besides a promoter located upstream of the glpE start codon, three internal glpGR promoters were identified that express glpG and/or glpR (glp repressor). One promoter was located just upstream of the glpG start codon and two others (separated by several hundred base pairs) were located within glpG upstream of the glpR start codon. The transcriptional start points of these promoters were identified by primer extension analysis. The strengths of the individual promoters were compared by analysis of their expression when fused to a pormoter-probe vector. Analysis of the transcriptional expression of the glpEGR sequence with different combinations of the glpEGR promoters revealed no internal transcriptional terminators within the entire operon. Thus, the glpEGR genes are co-transcribed and form a single complex operon. The presence of multiple promoters may provide for differential expression of glpE, glpG and glpR. Potential regulation of the operon promoters by GlpR, catabolite repression, anaerobiosis or by FIS was studied. The glpE promoter was apparently controlled by the cAMP-CRP complex, but none of the promoters was responsive to specific repression by GlpR, to anaerobiosis or to FIS. Specific repression exerted by GlpR was characterized in vivo using glpD-lacZ and glpK-lacZ fusions. The degree of repression was correlated with the level of GlpR expression, and was inefficient when the glpD-encoded glycerol-P dehydrogenase was absent, presumably due to accumulation of the inducer, glycerol-P. This is in contrast to the previous conclusion that gpsA-encoded glycerol-P synthase tightly controls the cellular level of glycerol-P by end product inhibition.

Biochemical and Genetic Characterization of PspE and GlpE, Two Single-domain Sulfurtransferases of Escherichia coli

The pspE and glpE genes of Escherichia coli encode periplasmic and cytoplasmic single-domain rhodaneses, respectively, that catalyzes sulfur transfer from thiosulfate to thiophilic acceptors. Strains deficient in either or both genes were constructed. Comparison of rhodanese activity in these strains revealed that PspE provides 85% of total rhodanese activity, with GlpE contributing most of the remainder. PspE activity was four times higher during growth on glycerol versus glucose, and was not induced by conditions that induce expression of the psp regulon. The glpE/pspE mutants displayed no apparent growth phenotypes, indicating that neither gene is required for biosynthesis of essential sulfur-containing molecules. PspE was purified by using cation exchange chromatography. Two distinct active peaks were eluted and differed in the degree of stable covalent modification, as assessed by mass spectrometry. The peak eluting earliest contained the equivalent mass of two additional sulfur atoms, whereas the second peak contained mainly one additional sulfur. Kinetic properties of purified PspE were consistent with catalysis occurring via a double-displacement mechanism via an enzyme-sulfur intermediate involving the active site cysteine. Kms for SSO32- and CN- were 2.7 mM and 32 mM, respectively, and kcat was 64s-1. The enzyme also catalyzed transfer of sulfur from thiosulfate to dithiothreitol, ultimately releasing sulfide.