Amino acid biosynthetic enzymes as targets of herbicide action

Several structurally-unrelated herbicides act by blocking amino acid biosynthesis. Since amino acid metabolism is similar in plants and well-studied, manipulatable microbial systems, the opportunity exists for a particularly productive interaction between microbial and plant molecular biology. Such a symbiosis may lead to new methods for the identification and design of crop protection chemicals.     

Characterization of the L-lysine biosynthetic pathway in the obligate methylotroph Methylophilus methylotrophus

The L-lysine biosynthetic pathway of the gram-negative obligate methylotroph Methylophilus methylotrophus AS1 was examined through characterization of the enzymes aspartokinase (AK), aspartsemialdehyde dehydrogenase, dihydrodipicolinate synthase (DDPS), dihydrodipicolinate reductase, and diaminopimelate decarboxylase. The AK was inhibited by L-threonine and by a combination of L-threonine and L-lysine, but not by L-lysine alone, and the activity of DDPS was moderately reduced by L-lysine. In an L-lysine producing mutant (G49), isolated as an S-(2-aminoethyl)-L-cysteine (lysine analog) resistant strain, both AK and DDPS were partially resistant to feedback inhibition. The ask and dapA genes encoding AK and DDPS respectively were isolated from the parental strain, AS1, and its G49 derivative. Comparison of the sequences revealed a point mutation in each of these genes in G49. The mutation in the ask gene altered aspartic acid in a key region involved in the allosteric regulation common to AKs, while a novel mutation in the dapA gene altered tyrosine-106, which was assumed to be involved in the binding of L-lysine to DDPS.     

L-Lysine biosynthetic pathway of Methylophilus methylotrophus and construction of an L-lysine producer

Previously, we showed that the enzymes aspartokinase (AK) and dihydrodipicolinate synthase (DDPS), which are involved in L-lysine biosynthesis in the Gram-negative obligate methylotroph Methylophilus methylotrophus AS1, were inhibited by allosteric effectors, including L-lysine. To elucidate further the regulation of L-lysine biosynthesis in M. methylotrophus, we cloned the genes encoding three other enzymes involved in this pathway, L-aspartate-beta-semialdehyde dehydrogenase, dihydrodipicolinate reductase (DDPR) and diaminopimelate decarboxylase, and examined their properties. DDPR was markedly inhibited by L-lysine. Based on this and our previous results, we constructed an L-lysine-producing strain of M. methylotrophus by introducing well-characterized genes encoding desensitized forms of AK and DDPS, as well as dapB (encoding DDPR) from Escherichia coli, using a broad host range plasmid. L-Lysine production was significantly increased by employing an S-(2-aminoethyl)-L-cysteine (L-lysine analog)-resistant mutant as the host. This derivative accumulated L-lysine at a concentration of 1 g l(-1) of medium using methanol as a carbon source.     

The molecular architecture of major enzymes from ajmaline biosynthetic pathway

The biosynthetic pathway leading to the monoterpenoid indole alkaloid ajmaline in Rauvolfia serpentiin serpentina is one of the most studied in the field of natural product biosynthesis. Ajmaline has a complex structure which is based on a six-membered ring system harbouring nine chiral carbon atoms. There are about fifteen enzymes involved, including some involving the side reactions of the ajmaline biosynthetic pathway. All enzymes exhibit pronounced substrate specificity. In the recent years isolation and sequencing of their cDNAs has allowed a detailed sequence analysis and comparison with functionally related and occasionally un-related enzymes. Site-directed mutations of several of the ajmaline-synthesizing enzymes have been performed and their catalytic residues have been identified. Success with over-expression of the enzymes was an important step for their crystallization and structural analysis by X-ray crystallography. Crystals with sufficient resolution were obtained from the major enzymes of the pathway. Strictosidine synthase has a 3D-structure with a six-bladed β-propeller fold the first time such a fold found in the plant kingdom. Its ligand complexes with tryptamine and secologanin, as well as structure-based sequence alignment, indicate a possible evolutionary relationship to several primary sequence-unrelated structures with this fold. The structure of strictosidine glucosidase was determined and its structure has as a (β/α)₈ barrel fold. Vinorine synthase provides the first 3D structure of a member of BAHD enzyme super-family. Raucaffricine glucosidase involved in a side-route of ajmaline biosynthesis has been crystallized. The ajmaline biosynthetic pathway is an outstanding example where many enzymes 3D-structure have been known and where there is a real potential for protein engineering to yield new alkaloid.     

Continuous spectrophotometric assays for three regulatory enzymes of the arginine biosynthetic pathway

N-Acetylglutamate synthase (AGS), N-acetylglutamate kinase (AGK), and glutamate N-acetyltransferase (GAT) are the key enzymes in the synthesis of arginine that serves as an important precursor for the synthesis of protein, polyamines, urea, and nitric oxide. Current assays available for these three enzymes are laborious and time-consuming and do not allow continuous monitoring of enzyme activities. Here we established continuous enzyme assays for AGS, AGK, and GAT based on the coupling of AGS and GAT reactions to AGK followed by coupling of the AGK reaction to N-acetylglutamate 5-phosphate reductase (AGPR). The rate of AGPR-dependent oxidation of reduced nicotinamide adenine dinucleotide phosphate was monitored continuously as a change in absorbance at 340 nm using spectrophotometry. These methods were applied to kinetic analyses for Escherichia coli AGK, E. coli AGS, and Saccharomyces cerevisiae GAT, and the kinetic parameters obtained in the coupling assays showed nearly the same values as those obtained previously using discontinuous assays. The specificity of these coupled assays was confirmed by the lack of enzyme activity from extracts of E. coli AGS-, E. coli AGK-, and S. cerevisiae GAT-deletion mutants. Moreover, the coupled assay enabled us to measure AGS activity from mammalian liver mitochondrial extracts, known to be an important regulatory enzyme for the urea cycle. These coupled enzyme assays are rapid, highly sensitive, and reproducible.     

Repression and derepression of the enzymes of the pyrimidine biosynthetic pathway in Salmonella typhimurium

Activities of five enzymes of the pyrimidine biosynthetic pathway and one enzyme involved in arginine synthesis were measured during batch culture of Salmonella typhimurium. Aspartate carbamoyltransferase, dihydroorotase, and the arginine pathway enzyme, ornithine carbamoyltransferase, remained constant during the growth cycle but showed a sharp decrease in activity after entering the stationary phase. Dihydroorotate dehydrogenase, orotate phosphoribosyltransferase and orotidine-5'-monophosphate (OMP) decarboxylase showed peaks of activity corresponding to the mid-point of the exponential phase of growth while remaining comparatively stable in the stationary phase. Derepression studies carried out by starving individual pyrimidine (Pyr-) deletion mutants for uracil showed that the extent of derepression obtained for aspartate carbamoyltransferase, dihydroorotase and dihydroorotate dehydrogenase depended on the location of the pyr gene mutation. Orotate phosphoribosyltransferase and OMP decarboxylase derepression levels were independent of the location of the pyr mutation. Aspartate carbamoyltransferase showed the greatest degree of derepression of the six enzymes studied, with pyrA strains (blocked in the first step of the pathway) showing about twice as much derepression as pyrF strains (blocked in the sixth step of the pathway). A study of the kinetics of repression on derepressed levels of the pyrimidine enzymes produced data that were compatible with dilution of specific activity by cell division when repressive amounts of uracil were added to the derepression medium.     

The structure of L-tyrosine 2,3-aminomutase from the C-1027 enediyne antitumor antibiotic biosynthetic pathway

The SgcC4 l-tyrosine 2,3-aminomutase (SgTAM) catalyzes the formation of (S)-beta-tyrosine in the biosynthetic pathway of the enediyne antitumor antibiotic C-1027. SgTAM is homologous to the histidine ammonia lyase family of enzymes whose activity is dependent on the methylideneimidazole-5-one (MIO) cofactor. Unlike the lyase enzymes, SgTAM catalyzes additional chemical transformations resulting in an overall stereospecific 1,2-amino shift in the substrate l-tyrosine to generate (S)-beta-tyrosine. Previously, we provided kinetic, spectroscopic, and mutagenesis data supporting the presence of MIO in the active site of SgTAM [Christenson, S. D.; Wu, W.; Spies, A.; Shen, B.; and Toney, M. D. (2003) Biochemistry 42, 12708-12718]. Here we report the first X-ray crystal structure of an MIO-containing aminomutase, SgTAM, and confirm the structural homology of SgTAM to ammonia lyases. Comparison of the structure of SgTAM to the l-tyrosine ammonia lyase from Rhodobacter sphaeroides provides insight into the structural basis for aminomutase activity. The results show that SgTAM has a closed active site well suited to retain ammonia and minimize the formation of lyase elimination products. The amino acid determinants for substrate recognition and catalysis can be predicted from the structure, setting the framework for detailed mechanistic investigations.     

Tryptophan biosynthetic pathway in the Enterobacteriaceae: some physical properties of the enzymes.

Several physical properties of the first four enzymatic activities of the tryptophan pathway were examined using gel filtration and ion exchange chromatography. Five different patterns were noted. Differences in the anthranilate synthetase (AS) and phosphoribosylanthranilate transferase (PRT) defined these patterns. In all the organisms studied phosphoribosylanthranilate isomerase and indoleglycerol phosphate synthetase co-eluted from both diethylaminoethyl-cellulose and G-200 and thus probably are contained in a single polypeptide of 50,000 daltons. An AS-PRT complex was found in Citrobacter species, Enterobacter cloacae, and Erwinia dissolvens. In all the other bacteria examined AS and PTR were separate molecules. In Serratia marcescens, S. marinorubra, and Enterobacter liquefaciens, AS was 140,000 daltons and PRT was 45,000 daltons. In Erwinia carotavora and Enterobacter hafniae the AS was the same size as the Serratia species but the PRT was larger at 67,000 daltons. Two Proteus species had an AS and PRT of the same size as E. carotavora and E. halfniae but the Proteus AS was different in that it partially dissociated upon gel filtration. Aeromonas formicans was unique in its possession of an AS with a molecular weight of 220,000. The PRT of A. formicans was found to elute at 67,000 daltons. Possible paths of evolution of the tryptophan enzymes are discussed in terms of the results of this study. The results presented here are also considered with respect to existing taxonomic schemes of the enteric bacteria.     

The heme biosynthetic pathway in the regenerating rat liver. The relation between enzymes of heme synthesis and growth

Enzymes of heme synthesis, porphyrins and heme content of regenerating rat livers were examined. During the first three days of regeneration the weights of livers of one-third and two-third hepatectomized rats increased 1.5-fold and 2.7-fold and the activity of porphobilinogen deaminase increased 2-fold and 4-fold and was inversely correlated with ferrochelatase activity. delta-Aminolevulinic acid synthase and delta-aminolevulinic acid dehydratase activities were reduced. Concomitantly an increase in the concentration of porphyrins and a decrease in that of heme were observed. The changes in the biosynthetic pathway of heme during rapid growth of the liver are discussed.     

Localization of two enzymes of the tetrahydrobiopterin biosynthetic pathway in embryonic chick retina.

Tetrahydrobiopterin (BH4) is an essential co-factor for the biosynthesis of catecholamine-type neurotransmitters and of nitric oxide (NO). The expression of the enzymes catalyzing the first two steps of the BH4 biosynthetic pathway was studied in the developing chicken retina by in situ hybridization and immunocytochemistry. GTP-cyclohydrolase-I (GTP-CH-I) and 6-pyruvoyl-tetrahydropterin synthase (PTPS) were already expressed in the undifferentiated and proliferating retina of E7. At stage E11 both enzymes were expressed in photoreceptors, amacrine cells, displaced amacrine cells, and ganglion cells, and in the plexiform layers in which synaptic connections take place. At stage E18 the labeling was comparable to E11 but appeared to be more concentrated in photoreceptors and ganglion cells.     

Mevalonate analogues as substrates of enzymes in the isoprenoid biosynthetic pathway of Streptococcus pneumoniae

Survival of the human pathogen Streptococcus pneumoniae requires a functional mevalonate pathway, which produces isopentenyl diphosphate, the essential building block of isoprenoids. Flux through this pathway appears to be regulated at the mevalonate kinase (MK) step, which is strongly feedback-inhibited by diphosphomevalonate (DPM), the penultimate compound in the pathway. The human mevalonate pathway is not regulated by DPM, making the bacterial pathway an attractive antibiotic target. Since DPM has poor drug characteristics, being highly charged, we propose to use unphosphorylated, cell-permeable prodrugs based on mevalonate that will be phosphorylated in turn by MK and phosphomevalonate kinase (PMK) to generate the active compound in situ. To test the limits of this approach, we synthesized a series of C₃-substituted mevalonate analogues to probe the steric and electronic requirements of the MK and PMK active sites. MK and PMK accepted substrates with up to two additional carbons, showing a preference for small substituents. This result establishes the feasibility of using a prodrug strategy for DPM-based antibiotics in S. pneumoniae and identified several analogues to be tested as inhibitors of MK. Among the substrates accepted by both enzymes were cyclopropyl, vinyl, and ethynyl mevalonate analogues that, when diphosphorylated, might be mechanism-based inactivators of the next enzyme in the pathway, diphosphomevalonate decarboxylase.     

Characterization of enzymes of the branched-chain amino acid biosynthetic pathway in Methanococcus spp.

Methanococcus aeolicus, Methanococcus maripaludis, and Methanococcus voltae contain similar levels of four enzymes of branched-chain amino acid biosynthesis: acetohydroxy acid synthase, acetohydroxy acid isomeroreductase, dihydroxy acid dehydratase, and transaminase B. Following growth at low partial pressures of H2-CO2, the levels of these enzymes in extracts of M. voltae are reduced three- to fivefold, which suggests that their synthesis is regulated. The enzymes from M. aeolicus were found to be similar to the eubacterial and eucaryotic enzymes with respect to molecular weights, pH optima, kinetic properties, and sensitivities to O2. The acetohydroxy acid isomeroreductase has a specific requirement for Mg2+, and other divalent cations were inhibitory. It was stimulated threefold by K+ and NH4+ ions and was able to utilize NADH as well as NADPH. The partially purified enzyme was not sensitive to O2. The dihydroxy acid dehydratase is extremely sensitive to O2, and it has a half-life under 5% O2 of 6 min at 25 degrees C. Divalent cations were required for activity, and Mg2+, Mn2+, Ni2+, Co2+, and Fe2+ were nearly equally effective. In conclusion, the archaebacterial enzymes are functionally homologous to the eubacterial and eucaryotic enzymes, which implies that this pathway is very ancient.     

Inactivation and activation of various membranal enzymes of the cholesterol biosynthetic pathway by digitonin

The activity of rat liver microsomal squalene epoxidase is inhibited effectively by digitonin. Concentrations of 0.8 to 1.2 mg/ml of digitonin cause total inhibition of microsomal (0.75 mg protein/ml) squalene epoxidase either in microsomes that were pretreated with digitonin and subsequently washed and subjected to epoxidase assay or when digitonin was added directly to the assay. The inhibition of squalene epoxidase by digitonin is concentration-dependent and takes place rapidly within 5 min of exposure of the microsomes to digitonin. Octylglucoside, dimethylsulfoxide, CHAPS, as well as cholesterol or total microsomal lipid extract were ineffective in restoring the digitonin-inhibited squalene epoxidase activity. Epoxidase activity in digitonin-treated microsomes was fully restored by Triton X-100. The reactivation by Triton X-100 displays a concentration optimum with maximal reactivation of the epoxidase (0.7 mg protein/ml) occurring at 0.2% Triton X-100. Microsomal 2,3-oxidosqualene-lanosterol cyclase is also inhibited by digitonin. Higher concentrations of digitonin are required to obtain full inhibition of the cyclase activity and only 40% inhibition of cyclase activity is observed at 1 mg/ml of digitonin. Solubilized (subunit size 55 to 66 kDa) and microsomal (subunit size 97 kDa) 3-hydroxy-3-methylglutaryl CoA reductase are totally unaffected by the same concentration of digitonin. Squalene synthetase, another microsomal enzyme in the biosynthetic pathway of cholesterol, is activated by digitonin. A 2.2-fold activation of squalene synthetase is observed at 0.8 mg/ml of digitonin. The results agree with a model in which squalene, and to a lesser degree 2,3-oxidosqualene, are segregated by digitonin into separate intramembranal pools.(ABSTRACT TRUNCATED AT 250 WORDS)     

Copper-induced oxidative stress in Saccharomyces cerevisiae targets enzymes of the glycolytic pathway

Increased cellular levels of reactive oxygen species are known to arise during exposure of organisms to elevated metal concentrations, but the consequences for cells in the context of metal toxicity are poorly characterized. Using two-dimensional gel electrophoresis, combined with immunodetection of protein carbonyls, we report here that exposure of the yeast Saccharomyces cerevisiae to copper causes a marked increase in cellular protein carbonyl levels, indicative of oxidative protein damage. The response was time dependent, with total-protein oxidation peaking approximately 15 min after the onset of copper treatment. Moreover, this oxidative damage was not evenly distributed among the expressed proteins of the cell. Rather, in a similar manner to peroxide-induced oxidative stress, copper-dependent protein carbonylation appeared to target glycolytic pathway and related enzymes, as well as heat shock proteins. Oxidative targeting of these and other enzymes was isoform-specific and, in most cases, was also associated with a decline in the proteins' relative abundance. Our results are consistent with a model in which copper-induced oxidative stress disables the flow of carbon through the preferred glycolytic pathway, and promotes the production of glucose-equivalents within the pentose phosphate pathway. Such re-routing of the metabolic flux may serve as a rapid-response mechanism to help cells counter the damaging effects of copper-induced oxidative stress.     

Reconstitution of the two terminal enzymes of the heme biosynthetic pathway into phospholipid vesicles

Purified mouse protoporphyrinogen oxidase (EC 1.3.3.4) and ferrochelatase (EC 4.99.1.1), the two terminal enzymes of the heme biosynthetic pathway, have been reconstituted into phospholipid vesicles, and the kinetics of the enzymes in the reconstituted systems were compared with the values obtained with the free enzymes. The apparent Km for free protoporphyrinogen oxidase in detergent solution is 5.61 +/- 0.62 microM for free protoporphyrinogen. The Km was lower when the enzyme was inserted into phospholipid vesicles (0.78 +/- 0.28 microM) and when both enzyme and substrate were incorporated into phospholipid vesicles (0.61 +/- 0.14 microM). In the presence of cardiolipin, a phospholipid present mainly in the inner mitochondrial membrane, the value of the Km for the substrate decreased 3-fold (0.20 +/- 0.02 microM). For reconstituted ferrochelatase similar kinetic analyses were carried out and it was found that the apparent Km values were only weakly affected by the lipid environment. Studies on the orientation of ferrochelatase demonstrated that approximately 50% of the enzyme in the reconstituted system had the active site located in the inner face of the phospholipid vesicle. This is in contrast to intact mitochondria where the active site is located on the matrix side of the inner mitochondrial membrane. The activation energies for both enzymes were determined for free and reconstituted enzymes. It was found that for both enzymes the activation energies were lower for the reconstituted systems than for the free enzymes.