Cloning and overproduction of biodegradative threonine deaminase from Escherichia coli W strain.

We have cloned the structural gene (tdcB) of biodegradative threonine deaminase from Escherichia coli W strain by utilizing the polymerase chain reaction. The JM109/pUCTDA strain, which was obtained by transforming E. coli JM109 with a vector plasmid (pUCTDA) containing the cloned tdcB gene, produced a large amount of the enzyme corresponding to more than 5% of the total soluble protein. Amino acid sequence analysis of this recombinant enzyme showed that the amino acid sequence is identical to the nucleotide-deduced sequence of biodegradative threonine deaminase from E. coli K-12.     

Cysteine and growth inhibition of Escherichia coli: threonine deaminase as the target enzyme.

Cysteine has been shown to inhibit growth in Escherichia coli strains C6 and HfrH 72, but not M108A. Growth inhibition was overcome by inclusion of isoleucine, leucine, and valine in the medium. Isoleucine biosynthesis was apparently affected, since addition of this amino acid alone could alter the inhibitory effects of cysteine. Homocysteine, mercaptoethylamine, and mercaptoethanol inhibited growth to varying degrees in some strains, these effects also being prevented by addition of branched-chain amino acids. Cysteine, mercaptoethylamine, and homocysteine were inhibitors of threonine deaminase but not transaminase B, two enzymes of the ilvEDA operon. Cysteine inhibition of threonine deaminase was reversed by threonine, although the pattern of inhibition was mixed. These results suggest a relationship between the growth-inhibitory effects of cysteine and other sulfur compounds and the inhibition of isoleucine synthesis at the level of threonine deaminase.     

Crystal structures of Salmonella typhimurium biodegradative threonine deaminase and its complex with CMP provide structural insights into ligand-induced oligomerization and enzyme activation

Two different pyridoxal 5'-phosphate-containing l-threonine deaminases (EC 4.3.1.19), biosynthetic and biodegradative, which catalyze the deamination of l-threonine to alpha-ketobutyrate, are present in Escherichia coli and Salmonella typhimurium. Biodegradative threonine deaminase (TdcB) catalyzes the first reaction in the anaerobic breakdown of l-threonine to propionate. TdcB, unlike the biosynthetic threonine deaminase, is insensitive to l-isoleucine and is activated by AMP. In the present study, TdcB from S. typhimurium was cloned and overexpressed in E. coli. In the presence of AMP or CMP, the recombinant enzyme was converted to the tetrameric form accompanied by significant enzyme activation. To provide insights into ligand-mediated oligomerization and enzyme activation, crystal structures of S. typhimurium TdcB and its complex with CMP were determined. In the native structure, TdcB is in a dimeric form, whereas in the TdcB.CMP complex, it exists in a tetrameric form with 222 symmetry and appears as a dimer of dimers. Tetrameric TdcB binds to four molecules of CMP, two at each of the dimer interfaces. Comparison of the dimer structure in the ligand (CMP)-free and -bound forms suggests that the changes induced by ligand binding at the dimer interface are essential for tetramerization. The differences observed in the tertiary and quaternary structures of TdcB in the absence and presence of CMP appear to account for enzyme activation and increased binding affinity for l-threonine. Comparison of TdcB with related pyridoxal 5'-phosphate-dependent enzymes points to structural and mechanistic similarities.     

Identification of lysyl residues at the AMP-binding site of biodegradative threonine deaminase from Escherichia coli.

The biodegradative threonine deaminase from Escherichia coli is activated allosterically by AMP. To identify the residues interacting with the phosphate group of AMP at the binding site, we used the affinity labeling reagent, adenosine diphosphopyridoxal (AP2-PL). In the absence of AMP, the enzyme formed the Schiff base with AP2-PL and Scatchard plot analysis showed a biphasic pattern, the respective Kd values for the high- and low-affinity binding phases being 20 and 110 microM. The former value is comparable to the Kd value of the enzyme for AMP. In the presence of AMP, the Schiff base formation was greatly reduced. Although the maximal activating effect of adenosine diphosphopyridoxine, a non-reactive derivative of AP2-PL, was about 13% of that of AMP, the half-saturation concentration was almost the same. These findings suggest that AP2-PL specifically labeled the lysyl residue(s) at the AMP-binding site of the enzyme. To identify the labeled residue(s), we reduced the modified enzyme with sodium borohydride, then cleaved it with cyanogen bromide and Achromobacter lyticus protease I. Reverse-phase HPLC was used to isolate two labeled peptides from the digest. Their amino acid compositions and sequences showed that Lys-111 and Lys-113 were labeled. We conclude that these two lysyl residues are located around the phosphate group of AMP at the allosteric regulation site of the enzyme.     

Feedback inhibition of homoserine dehydrogenase and threonine deaminase in the genusBifidobacterium

Feedback inhibition of crude and purified extracts of homoserine dehydrogenase and threonine deaminase activities in the genusBifidobacterium was studied. Homoserine dehydrogenase was partially or completely inhibited byl-threonine, and a marked inhibitory effect byl-isoleucine on threonine deaminase was observed. In the speciesBifidobacterium cuniculi high levels ofl-valine reversed the inhibitory effect ofl-isoleucine. The -aminobutyric acid-resistant mutant Ru 326/106 of the speciesB. ruminale, overproducer ofl-isoleucine, had a derepressed homoserine dehydrogenase and a lesser feedback inhibition byl-threonine. Homoserine dehydrogenase appeared to be in bifids specifically NAD dependent. The regulatory mechanisms of aspartate family amino acid biosynthesis in bifidobacteria was discussed.     

Regulation of cell proliferation and morphogenesis by amino acids inBrassica tissue cultures and its correlation with threonine deaminase

The effect of amino acids has been investigated with respect to the capacity ofBrassica cultures to undergo proliferation and differentiation. Hormone medium without any amino acid resulted in 6% shoot formation. Addition of optimal concentrations of L-leucine and L-isoleucine enhanced shoot formation upto 30% and 60%, respectively. L-methionine, L-threonine and pyruvic acid supported only proliferation but no differentiation. Amino acids had a marked effect on the activity of enzyme threonine deaminase (TD), bothin vivo andin vitro. TD in proliferating callus cultures was 3-fold higher than in differentiating cultures. Amino acids which induced cell proliferation increased TD while those which supported differentiation repressed it. Amino acids which did not alter TD activity had no effect on morphogenesis. The results suggest that amino acids play a regulatory role inBrassica morphogenesis which can be correlated with the activity of threonine deaminase.     

The design and synthesis of inhibitors of adenosine 5'-monophosphate deaminase.

Carbocylic coformycin (4) is a potent herbicide whose primary mode of action involves inhibition of adenosine 5'-monophosphate deaminase (AMPDA) following phosphorylation of the 5'-hydroxyl group in vivo. The search for more stable and accessible structures led to the synthesis of carbocyclic nebularine (8) and deaminoformycin (10). The latter compound is a good herbicide and its corresponding 5'-monophosphate 14 is a strong inhibitor of plant AMPDA (IC50 100 nM).     

Regulation of skeletal-muscle AMP deaminase. Evidence for a highly pH-dependent inhibition by ATP of the homogeneous derivative of the rabbit enzyme yielded by limited proteolysis.

Limited proteolysis of rabbit skeletal-muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) with trypsin results in conversion of the enzyme into a species which over the pH range 6.5-7.1 exhibits hyperbolic kinetics at low K+ concentration even in the absence of ADP, but shows a 20% decrease in activity at saturating substrate concentration. Analysis by sedimentation-equilibrium techniques reveals the proteolysed enzyme to be homogeneous and to have a molecular mass of 222,000 Da, indicative of a trimeric structure with a subunit molecular mass of 72,000 Da, in contrast with the tetrameric structure of the native enzyme, composed of four 79,000-Da subunits. These observations suggest a role of the 7,000-Da fragment which is removed by proteolysis in the maintenance of the three-dimensional structure of the subunit that causes the enzyme at low K+ concentration to show homotropic positive co-operativity. Study of the influence of pH, isolated from that of K+, on the kinetics of AMP deaminase reveals a highly pH-dependent inhibitory effect by ATP which is completely absent at acid pH values and abruptly manifests itself just above neutrality. This phenomenon may have significance in the metabolism of exercising muscle, in connection with the pH-dependent interaction of AMP deaminase with the thick filament.