A novel alpha-ketoglutarate reductase activity of the serA-encoded 3-phosphoglycerate dehydrogenase of Escherichia coli K-12 and its possible implications for human 2-hydroxyglutaric aciduria.

Escherichia coli serA-encoded 3-phosphoglycerate (3PG) dehydrogenase catalyzes the first step of the major phosphorylated pathway of L-serine (Ser) biosynthesis. The SerA enzyme is evolutionarily related to the pdxB gene product, 4-phosphoerythronate dehydrogenase, which catalyzes the second step in one branch of pyridoxal 5'-phosphate coenzyme biosynthesis. Both the Ser and pyridoxal 5'-phosphate biosynthetic pathways use the serC(pdxF)-encoded transaminase in their next steps. In an analysis of these parallel pathways, we attempted to couple the transaminase and dehydrogenase reactions in the reverse direction. Unexpectedly, we found that the SerA enzyme catalyzes a previously undetected reduction of alpha-ketoglutarate (alpha KG) to 2-hydroxyglutaric acid (HGA). Numerous criteria ruled out the possibility that this SerA alpha KG reductase activity was caused by contamination in the substrate or purified enzyme preparations. HGA was confirmed as the product of the SerA alpha KG reductase reaction by thin-layer chromatography and by enzyme assays showing that both the D- and L-isomers of HGA were substrates for the reverse (dehydrogenase) reaction. Detailed steady-state kinetic analyses showed that alpha KG reduction (apparent Michaelis-Menten constant [Km(app)] = 88 microM; apparent catalytic constant [kcat(app)] = 33.3 s-1) and 3-phosphohydroxypyruvate reduction (Km(app) = 3.2 microM; kcatapp = 27.8 s-1), which is the reverse reaction of 3PG oxidation, were the major in vitro activities of the SerA enzyme. The SerA alpha KG reductase was inhibited by Ser, D-HGA, 3PG, and glycine (Gly), whereas the D-HGA dehydrogenase was inhibited by Ser, alpha KG, 3-phosphohydroxypyruvate, and Gly. The implications of these findings for the regulation of Ser biosynthesis, the recycling of NADH, and the enzymology of 2-hydroxyacid dehydrogenases are discussed. Since the same pathway of Ser biosynthesis seems to be present in all organisms, these results suggest that a mutation in the human SerA homolog may contribute to the neurometabolic diseases D- and L-2-hydroxyglutaric aciduria, which lead to the accumulation of D-HGA and L-HGA, respectively.     

Suppression of insertions in the complex pdxJ operon of Escherichia coli K-12 by lon and other mutations.

Complementation analyses using minimal recombinant clones showed that all known pdx point mutations, which cause pyridoxine (vitamin B6) or pyridoxal auxotrophy, are located in the pdxA, pdxB, serC, pdxJ, and pdxH genes. Antibiotic enrichments for chromosomal transposon mutants that require pyridoxine (vitamin B6) or pyridoxal led to the isolation of insertions in pdxA, pdxB, and pdxH but not in pdxJ. This observation suggested that pdxJ, like pdxA, pdxB, and serC, might be in a complex operon. To test this hypothesis, we constructed stable insertion mutations in and around pdxJ in plasmids and forced them into the bacterial chromosome. Physiological properties of the resulting insertion mutants were characterized, and the DNA sequence of pdxJ and adjacent regions was determined. These combined approaches led to the following conclusions: (i) pdxJ is the first gene in a two-gene operon that contains a gene, temporarily designated dpj, essential for Escherichia coli growth; (ii) expression of the rnc-era-recO and pdxJ-dpj operons can occur independently, although the pdxJ-dpj promoter may lie within recO; (iii) pdxJ encodes a 26,384-Da polypeptide whose coding region is preceded by a PDX box, and dpj probably encodes a basic, 14,052-Da polypeptide; (iv) mini-Mud insertions in dpj and pdxJ, which are polar on dpj, severely limit E. coli growth; and (v) three classes of suppressors, including mutations in lon and suppressors of lon, that allow faster growth of pdxJ::mini-Mud mutants can be isolated. A model to account for the action of dpj suppressors is presented, and aspects of this genetic analysis are related to the pyridoxal 5'-phosphate biosynthetic pathway.     

An unusual genetic link between vitamin B6 biosynthesis and tRNA pseudouridine modification in Escherichia coli K-12

We characterized several unusual phenotypes caused by stable insertion mutations in a gene that is located upstream in the same operon from hisT, which encodes the tRNA modification enzyme pseudouridine synthase I. Mutants containing kanamycin resistance (Kmr) cassettes in this upstream gene, which we temporarily designated usg-2, failed to grow on minimal plus glucose medium at 37 and 42 degrees C. However, usg-2::Kmr mutants did form oddly translucent, mucoid colonies at 30 degrees C or below. Microscopic examination revealed that cells from these translucent colonies were spherical and seemed to divide equatorially. Addition of D-alanine restored the shape of the mutant cells to rods and allowed the mutants to grow slowly at 37 degrees C and above. By contrast, addition of the common L-amino acids prevented growth of the usg-2::Kmr mutants, even at 30 degrees C. Furthermore, prolonged incubation of usg-2::Kmr mutants at 37 and 42 degrees C led to the appearance of several classes of temperature-resistant pseudorevertants. Other compounds also supported growth of usg-2::Kmr mutants at 37 and 42 degrees C, including glycolaldehyde and the B6 vitamers pyridoxine and pyridoxal. This observation suggested that usg-2 was pdxB, which had been mapped near hisT. Complementation experiments confirmed that usg-2 is indeed pdxB, and inspection of the pyridoxine biosynthetic pathway suggests explanations for the unusual phenotypes of pdxB::Kmr mutants. Finally, Southern hybridization experiments showed that pdxB and hisT are closely associated in several enterobacterial species. We consider reasons for grouping pdxB and hisT together in the same complex operon and speculate that these two genes play roles in the global regulation of amino acid metabolism.     

Pyridoxine-requiring mutants of Escherichia coli: glycolaldehyde dehydrogenase is not coded for by the pdxB gene.

Twenty-seven independent pyridoxineless mutants belonging to genetic linkage group I were assayed for glycolaldehyde dehydrogenase. Some mutants lacked enzyme activity entirely, and others showed activity ranging from very low to wild-type levels. Reversion to pyridoxine independence usually had no effect upon this activity. Transfer of the pyridoxine genes to a common host that had wild-type levels of enzyme activity made the recipient pyridoxineless without affecting the activity. These results negate the idea of an obligatory role for glycolaldehyde dehydrogenase in pyridoxine biosynthesis.     

Biochemical characterization of gapB-encoded erythrose 4-phosphate dehydrogenase of Escherichia coli K-12 and its possible role in pyridoxal 5'-phosphate biosynthesis.

One step in de novo pyridoxine (vitamin B6) and pyridoxal 5'-phosphate biosynthesis was predicted to be an oxidation catalyzed by an unidentified D-erythrose-4-phosphate dehydrogenase (E4PDH). To help identify this E4PDH, we purified the Escherichia coli K-12 gapA- and gapB-encoded dehydrogenases to homogeneity and tested whether either uses D-erythrose-4-phosphate (E4P) as a substrate. gapA (gap1) encodes the major D-glyceraldehyde-3-phosphate dehydrogenase (GA3PDH). The function of gapB (gap2) is unknown, although it was suggested that gapB encodes a second form of GA3PDH or is a cryptic gene. We found that the gapB-encoded enzyme is indeed an E4PDH and not a second GA3PDH, whereas gapA-encoded GA3PDH used E4P poorly, if at all, as a substrate under the in vitro reaction conditions used in this study. The amino terminus of purified E4PDH matched the sequence predicted from the gapB DNA sequence. Purified E4PDH was a heat-stable tetramer with a native molecular mass of 132 kDa. E4PDH had an apparent Km value for E4P [Kmapp(E4P)] of 0.96 mM, an apparent kcat catalytic constant for E4P [kcatapp(E4P)] of 200 s-1, Kmapp(NAD+) of 0.074 mM, and kcatapp(NAD+) of 169 s-1 in steady-state reactions in which NADH formation was determined. From specific activities in crude extracts, we estimated that there are at least 940 E4PDH tetramer molecules per bacterium growing in minimal salts medium plus glucose at 37 degrees C. Thin-layer chromatography confirmed that the product of the E4PDH reaction was likely the aldonic acid 4-phosphoerythronate. To establish a possible role of E4PDH in pyridoxal 5'-phosphate biosynthesis, we showed that 4-phosphoerythronate is a likely substrate for the 2-hydroxy-acid dehydrogenase encoded by the pdxB gene. Implications of these findings in the evolution of GA3PDHs are also discussed. On the basis of these results, we propose renaming gapB as epd (for D-erythrose-4-phosphate dehydrogenase).     

Analysis of the genes flanking xabB: a methyltransferase gene is involved in albicidin biosynthesis in Xanthomonas albilineans

Transposon mutagenesis and complementation studies previously identified a gene (xabB) for a large (526kDa) polyketide-peptide synthase required for biosynthesis of albicidin antibiotics and phytotoxins in the sugarcane leaf scald pathogen Xanthomonas albilineans. A cistron immediately downstream from xabB encodes a polypeptide of 343aa containing three conserved motifs characteristic of a family of S-adenosyl-L-methionine (SAM)-dependent O-methyltransferases. Insertional mutagenesis and complementation indicate that the product of this cistron (designated xabC) is essential for albicidin production, and that there is no other required downstream cistron. The xabB promoter region is bidirectional, and insertional mutagenesis of the first open reading frame (ORF) in the divergent gene also blocks albicidin biosynthesis. This divergent ORF (designated thp) encodes a protein of 239aa displaying high similarity to several IS21-like transposition helper proteins. The thp cistron is not located in a recognizable transposon, and is probably a remnant from a past transposition event that may have contributed to the development of the albicidin biosynthetic gene cluster. Failure of 'in trans' complementation of thp indicates that a downstream cistron transcribed with thp is required for albicidin biosynthesis.     

Cloning and sequencing of the serine dehydrogenase gene from Agrobacterium tumefaciens

The structural gene for NADP+-dependent serine dehydrogenase [EC 1.1.1.-] from Agrobacterium tumefaciens ICR 1600 was cloned into Escherichia coli cells and its complete DNA sequence was analyzed. The gene encodes a polypeptide containing 249 amino acid residues. The enzyme had high sequence similarity to short-chain alcohol dehydrogenases from bacteria and unknown proteins of Haemophilus influenzae, Escherichia coli, and Saccharomyces cerevisiae.     

Nucleotide sequence of the Escherichia coli entE gene

The Escherichia coli entE gene encodes a polypeptide necessary in the latter stages of biosynthesis of the siderophore enterobactin. The entE gene and adjacent DNA were sequenced. The predicted EntE polypeptide consists of 536 amino acids and has a Mr of 58,299 and a net charge of -7.33. Genetic evidence combined with this and previous sequencing data indicate that the genes entCEB(G)A are transcribed as unit from a promoter upstream of entC.     

Cloning and sequence analysis of a glyceraldehyde-3-phosphate dehydrogenase gene from Ganoderma lucidum

A cDNA library of Ganoderma lucidum has been constructed using a Zap Express cloning vector. A glyceraldehyde-3-phosphate dehydrogenase gene (gpd) was isolated from this library by hybridization of the recombinant phage clones with a gpd-specific gene probe generated by PCR. By comparison of the cDNA and the genomic DNA sequences, it was found that the complete nucleotide sequence encodes a putative polypeptide chain of 338 amino acids interrupted by 6 introns. The predicted amino acid sequence of this gene shows a high degree of sequence similarity to the GPD proteins from yeast and filamentous fungi. The promoter region contains a CT-rich stretch, two CAAT boxes, and a consensus TATA box. The possibility of using the gpd promoter in the construction of new transformation vectors is discussed.     

Flavin adenine dinucleotide-dependent 4-phospho-D-erythronate dehydrogenase is responsible for the 4-phosphohydroxy-L-threonine pathway in vitamin B6 biosynthesis in Sinorhizobium meliloti

The vitamin B6 biosynthetic pathway in Sinorhizobium meliloti is similar to that in Escherichia coli K-12; in both organisms this pathway includes condensation of two intermediates, 1-deoxy-D-xylulose 5-phosphate and 4-phosphohydroxy-L-threonine (4PHT). Here, we report cloning of a gene designated pdxR that functionally corresponds to the pdxB gene of E. coli and encodes a dye-linked flavin adenine dinucleotide-dependent 4-phospho-D-erythronate (4PE) dehydrogenase. This enzyme catalyzes the oxidation of 4PE to 3-hydroxy-4-phosphohydroxy-alpha-ketobutyrate and is clearly different in terms of cofactor requirements from the pdxB gene product of E. coli, which is known to be an NAD-dependent enzyme. Previously, we revealed that in S. meliloti IFO 14782, 4PHT is synthesized from 4-hydroxy-l-threonine and that this synthesis starts with glycolaldehyde and glycine. However, in this study, we identified a second 4PHT pathway in S. meliloti that originates exclusively from glycolaldehyde (the major pathway). Based on the involvement of 4PE in the 4PHT pathway, the incorporation of different samples of 13C-labeled glycolaldehyde into pyridoxine molecules was examined using 13C nuclear magnetic resonance spectroscopy. On the basis of the spectral analyses, the synthesis of 4PHT from glycolaldehyde was hypothesized to involve the following steps: glycolaldehyde is sequentially metabolized to D-erythrulose, D-erythrulose 4-phosphate, and D-erythrose 4-phosphate by transketolase, kinase, and isomerase, respectively; and D-erythrose 4-phosphate is then converted to 4PHT by the conventional three-step pathway elucidated in E. coli, although the mechanism of action of the enzymes catalyzing the first two steps is different.