A chromosomal cluster of genes encoding ADP-glucose synthetase, glycogen synthase and phosphoglucomutase in Agrobacterium tumefaciens.

A chromosomal region from Agrobacterium tumefaciens that complements exoC (pgm) mutations was cloned and sequenced. A cluster of three open reading frames (ORF1, ORF2 and ORF3) was identified. These genes are oriented in the same direction and are involved in the synthesis of glycogen and other polysaccharides. ORF1 encodes a 420-amino-acid (aa) protein with 55.9% homology to Escherichia coli GlgC (ADP-glucose synthetase, EC 2.7.7.27). ORF2 encodes a 480-aa protein with 42.2% homology to E. coli GlgA (glycogen synthase, EC 2.4.1.21). Based on Tn5 mutagenesis and protein homology, ORF3 was identified as the structural gene encoding phosphoglucomutase (Pgm, EC 2.7.5.1). ORF3 encodes a 542-aa protein with 52.6% homology to rabbit Pgm. There is no significant homology (less than 20%) to the Xanthomonas campestris XanA protein, which displays phosphomannomutase (Pmm) and Pgm activities [Koplin et al., J. Bacteriol 174 (1992) 191-199]. An A. tumefaciens pgm::Tn5 mutant retains Pmm activity.

Purification and characterisation of a novel iso-propanol dehydrogenase from Phytomonas sp.

An alcohol dehydrogenase with two identical subunits and a subunit molecular mass of 40,000 was purified from Phytomonas sp. isolated from the lactiferous tubes of Euphorbia characias. Digitonin titration and subcellular fractionation suggest that the enzyme is present in the mitochondrion. It utilises as substrates, primary and secondary alcohols, is specific for NAD+ as coenzyme and is inhibited by HgCl(2). The pH optimum for the oxidation of ethanol is 9.5, and for the reverse reaction 8.5. The apparent Km values for iso-propanol and ethanol are 40 and 34 microM, respectively and for the reverse reaction, with acetone as substrate, 14 microM. The respective specific activities with iso-propanol and ethanol as substrate, as measured in crude extracts are 300 and 16 mU (milligram of protein)-1. In isoelectric focusing the enzyme showed three major bands with slightly differing isoelectric points that ranged from 6.4 to 6.8. The name, iso-propanol dehydrogenase is proposed for this enzyme.

Characterisation of the two malate dehydrogenases from Phytomonas sp. Purification of the glycosomal isoenzyme.

Two NAD(H)-dependent malate dehydrogenase (MDH) isoenzymes were detected in Phytomonas isolated from the lactiferous tubes of Euphorbia characias. The total specific activity in crude extracts using oxaloacetate as substrate was 3.3 U mg-1 of protein. The two isoenzymes had isoelectric points of 6.0 and 7.2, respectively. The acidic isoform represented 80% of the total activity in the cell and was present in the glycosome. It was purified to homogeneity by a method involving hydrophobic interaction chromatography on Phenyl-Sepharose followed by ionic exchange on CM-Sepharose and affinity chromatography on Blue-Sepharose. The purified glycosomal MDH is a homodimeric protein with a subunit molecular mass of 37 kDa and it has a low substrate specificity, since it was able to reduce both aromatic and aliphatic alpha-ketoacids as substrate including oxaloacetate, phenyl pyruvate, alpha-keto iso-caproate and pyruvate. The apparent K(m)s for oxaloacetate and NADH were 166 and 270 microM, respectively and for L-malate and NAD+, 3000 and 246 microM, respectively. The basic isoform was present in the mitochondrion. It has a high substrate specificity and an apparent K(m) of 132 and 63 microM for oxaloacetate and NADH, respectively, and of 450 and 91 microM, respectively, with L-malate and NAD+.

Cloning and expression of the glgC gene from Agrobacterium tumefaciens: purification and characterization of the ADPglucose synthetase.

The gene encoding ADPglucose synthetase (EC 2.7.7.27) from Agrobacterium tumefaciens was isolated and expressed in Escherichia coli. The recombinant protein was purified to electrophoretic homogeneity in steps including ion-exchange and hydrophobic chromatography. The same purification procedure was utilized to purify ADPglucose synthetase from A. tumefaciens cells. The enzymes from the two sources were purified and characterized and were found to have identical kinetic, regulatory, and structural properties. In polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, only one polypeptide band of 50 kDa was detected. In immunoblotting following electrophoresis, the 50-kDa band reacted with antibodies raised against the Escherichia coli ADPglucose synthetase; there was no reaction with antibodies raised against the spinach enzyme. The immunoreactivity of the A. tumefaciens ADPglucose synthetase was confirmed in antibody neutralization assays. Using gel filtration, the native enzyme was shown to be a tetramer. Fructose 6-phosphate and pyruvate were the most effective activators of the enzyme; maximal activation was observed in the ADPglucose synthesis direction, in which the enzyme was activated about ninefold by fructose 6-phosphate and fivefold by pyruvate. Both activators increased the affinity of the enzyme for the substrates ATP and glucose 1-phosphate. Inorganic orthophospate, ADP, AMP, and pyridoxal phosphate behaved as inhibitors of the enzyme. The distinctive regulatory properties of the enzyme from A. tumefaciens are compared with those of two enterobacterial enzymes and discussed in the context of their deduced amino acid sequences.

A family of highly conserved glycosomal 2-hydroxyacid dehydrogenases from Phytomonas sp.

Phytomonas sp. contains two malate dehydrogenase isoforms, a mitochondrial isoenzyme with a high specificity for oxaloacetate and a glycosomal isozyme that acts on a broad range of substrates (Uttaro, A. D., and Opperdoes, F.R. (1997) Mol. Biochem. Parasitol. 89, 51-59). Here, we show that the low specificity of the latter isoenzyme is the result of a number of recent gene duplications that gave rise to a family of glycosomal 2-hydroxyacid dehydrogenase genes. Two of these genes were cloned, sequenced, and overexpressed in Escherichia coli. Although both gene products have 322 amino acids, share 90.4% identical residues, and have a similar hydrophobicity profile and net charge, their kinetic properties were strikingly different. One isoform behaved as a real malate dehydrogenase with a high specificity for oxaloacetate, whereas the other showed no activity with oxaloacetate but was able to reduce other oxoacids, such as phenyl pyruvate, 2-oxoisocaproate, 2-oxovalerate, 2-oxobutyrate, 2-oxo-4-methiolbutyrate, and pyruvate.

Biochemical characterization of avirulent exoC mutants of Agrobacterium tumefaciens.

The synthesis of periplasmic beta(1-2)glucan is required for crown gall tumor formation by Agrobacterium tumefaciens and for effective nodulation of alfalfa by Rhizobium meliloti. The exoC (pscA) gene is required for this synthesis by both bacteria as well as for the synthesis of capsular polysaccharide and normal lipopolysaccharide. We tested the possibility that the pleiotropic ExoC phenotype is due to a defect in the synthesis of an intermediate common to several polysaccharide biosynthetic pathways. Cytoplasmic extracts from wild-type A. tumefaciens and from exoC mutants of A. tumefaciens containing a cloned wild-type exoC gene synthesized in vitro UDP-glucose from glucose, glucose 1-phosphate, and glucose 6-phosphate. Extracts from exoC mutants synthesized UDP-glucose from glucose 1-phosphate but not from glucose or glucose 6-phosphate. Membranes from exoC mutant cells synthesized beta(1-2)glucan in vitro when exogenous UDP-glucose was added and contained the 235-kilodalton protein, which has been shown to carry out this synthesis in wild-type cells. We conclude that the inability of exoC mutants to synthesize beta(1-2)glucan is due to a deficiency in the activity of the enzyme phosphoglucomutase (EC 2.7.5.1), which in wild-type bacteria converts glucose 6-phosphate to glucose 1-phosphate, an intermediate in the synthesis of UDP-glucose. This interpretation can account for all of the deficiencies in polysaccharide synthesis which have been observed in these mutants.

Pyruvate kinase of Trypanosoma brucei: overexpression, purification, and functional characterization of wild-type and mutated enzyme.

A procedure was developed for overexpression of Trypanosoma brucei pyruvate kinase in Escherichia coli. The enzyme was purified to near-homogeneity from the bacterial lysate by first removing nucleic acids and contaminating proteins by protamine sulfate precipitation and subsequent passage over a phosphocellulose column. The purified protein is essentially indistinguishable in its physicochemical and kinetic properties from the enzyme purified from trypanosomes. Furthermore, experiments were undertaken to locate the binding site of the allosteric effector fructose 2,6-bisphosphate. Regulation of pyruvate kinase by this effector is unique to trypanosomes and related protozoan organisms. Therefore, a three-dimensional structure model of the enzyme was made, and a putative effector-binding site could be identified in an interdomain cleft. Four residues in this cleft were mutated, and the mutant proteins were produced and purified, using the same methodology as for the wild-type pyruvate kinase. Some mutants showed only minor changes in the activation by the effector. However, substitution of Arg22 by Gly resulted in a 9.2-fold higher S(0.5) for phosphoenolpyruvate and a significantly smaller kcat than the wild-type enzyme. Furthermore, the apparent affinity of this mutant for the allosteric effectors fructose 1,6-bisphosphate and fructose 2,6-bisphosphate was 8.2- and 5.2-fold lower than that of its wild-type counterpart. Effector binding was also affected, although to a lesser extent, in a mutant Phe463Val. These data indicate that particularly residue Arg22, but also Phe463, are somehow involved in the binding of the allosteric effectors.