Remarkable Diversity in the Enzymes Catalyzing the Last Step in Synthesis of the Pimelate Moiety of Biotin

Biotin synthesis in Escherichia coli requires the functions of the bioH and bioC genes to synthesize the precursor pimelate moiety by use of a modified fatty acid biosynthesis pathway. However, it was previously noted that bioH has been replaced with bioG or bioK within the biotin synthetic gene clusters of other bacteria. We report that each of four BioG proteins from diverse bacteria and two cyanobacterial BioK proteins functionally replace E. coli BioH in vivo. Moreover, purified BioG proteins have esterase activity against pimeloyl-ACP methyl ester, the physiological substrate of BioH. Two of the BioG proteins block biotin synthesis when highly expressed and these toxic proteins were shown to have more promiscuous substrate specificities than the non-toxic BioG proteins. A postulated BioG-BioC fusion protein was shown to functionally replace both the BioH and BioC functions of E. coli. Although the BioH, BioG and BioK esterases catalyze a common reaction, the proteins are evolutionarily distinct.     

Indirect evidence for a strict negative control of S-adenosyl-l-methionine decarboxylase by spermidine in rat hepatoma cells

1. Direct or indirect inhibitors of l-ornithine decarboxylase (EC 4.1.1.17), structurally related or unrelated to l-ornithine, including dl-α-difluoromethylornithine, α-methylornithine and 1,3-diaminopropane, used alone or in combination, decreased polyamine concentrations in rat hepatoma tissue culture (HTC) cells and increased S-adenosyl-l-methionine decarboxylase activity (EC 4.1.1.50). 2. Comparison of the catalytic properties of S-adenosyl-l-methionine from cells with elevated and normal activities revealed no apparent modification of the catalytic site as judged by affinity for the substrate, stimulation by di- and tri-amines and inhibition by methylglyoxal bis-(guanylhydrazone). 3. Actinomycin D and cycloheximide, and RNA and a proteinsynthesis inhibitor respectively, blocked the increase of S-adenosyl-l-methionine decarboxylase activity elicited by α-difluoromethylornithine. In polyamine-depleted cells the apparent half-life of elevated S-adenosyl-l-methionine decarboxylase activity, determined by inhibition of protein synthesis, was 2.5-fold longer than in control cells. The present results suggest that elevation of S-adenosyl-l-methionine decarboxylase activity by α-difluoromethylornithine is due to stabilization of the enzyme. 4. Restoration of the normal intracellular putrescine content, by addition of putrescine to the medium of polyamine-deficient cells, transiently increased S-adenosyl-l-methionine decarboxylase activity. Thereafter, intracellular conversion of putrescine into spermidine was accompanied by inactivation of the enzyme at a rate that was similar to that found on addition of spermidine itself. No relationship between total intracellular spermine content and S-adenosyl-l-methionine decarboxylase activity could be established. 5. Addition of 1mm-1,3-diaminopropane to polyamine-deficient cells did not cause a decrease in the activity of S-adenosyl-l-methionine decarboxylase, whereas addition of 1,5-diaminopentane (cadaverine) did. 1,3-Diamino-N-(3-aminopropyl)propane did not accumulate in cells treated with α-difluoromethylornithine and 1,3-diaminopropane, whereas addition of 1,5-diaminopentane led to the accumulation of 1,5-diamino-N-(3-aminopropyl)pentane. 1,3-Diamino-N-(3-aminopropyl)propane (10μm) was as effective as spermidine in decreasing S-adenosyl-l-methionine decarboxylase activity. Thus effectiveness of a diamine in decreasing enzyme activity is related to its capability of being converted into a closely structurally related homologue of spermidine by spermidine synthase. 6. The spermidine site of action appears to be post-translational since (a) the spermidine-induced decrease of S-adenosyl-l-methionine activity was not prevented by actinomycin D and (b) spermidine in the presence of cycloheximide led to a synergistic inactivation of the enzyme with a decay rate that progressively approached control values. Altogether these results are indirect evidence for a strict negative control of S-adenosyl-l-methionine decarboxylase by spermidine and substantiate previous findings [Mamont, Duchesne, Grove & Tardif (1978) Exp. Cell Res. 115, 387–393]. Spermidine appears to act on some processes involved in denaturation and/or degradation of the enzyme protein. Putrescine appears to decrease the rate of these processes. The physiological significance of the regulatory control of S-adenosyl-l-methionine decarboxylase is discussed.     

Studies of inhibition of rat spermidine synthase and spermine synthase

1. S-Adenosyl-l-methionine, S-adenosyl-l-homocysteine, 5′-methylthioadenosine and a number of analogues having changes in the base, sugar or amino acid portions of the molecule were tested as potential inhibitors of spermidine synthase and spermine synthase from rat ventral prostate. 2. S-Adenosyl-l-methionine was inhibitory to these reactions, as were other nucleosides containing a sulphonium centre. The most active of these were S-adenosyl-l-ethionine, S-adenosyl-4-methylthiobutyric acid, S-adenosyl-d-methionine and S-tubercidinylmethionine, which were all comparable in activity with S-adenosylmethionine itself, producing 70–98% inhibition at 1mm concentrations. Spermine synthase was somewhat more sensitive than spermidine synthase. 3. 5′-Methylthioadenosine, 5′-ethylthioadenosine and 5′-methylthiotubercidin were all powerful inhibitors of both enzymes, giving 50% inhibition of spermine synthase at 10–15μm and 50% inhibition of spermidine synthase at 30–45μm. 4. S-Adenosyl-l-homocysteine was a weak inhibitor of spermine synthase and practically inactive against spermidine synthase. Analogues of S-adenosylhomocysteine lacking either the carboxy or the amino group of the amino acid portion were somewhat more active, as were derivatives in which the ribose ring had been opened by oxidation. The sulphoxide and sulphone derivatives of decarboxylated S-adenosyl-l-homocysteine and the sulphone of S-adenosyl-l-homocysteine were quite potent inhibitors and were particularly active against spermidine synthase (giving 50% inhibition at 380, 50 and 20μm respectively). 5. These results are discussed in terms of the possible regulation of polyamine synthesis by endogenous nucleosides and the possible value of some of the inhibitory substances in experimental manipulations of polyamine concentrations. It is suggested that 5′-methylthiotubercidin and the sulphone of S-adenosylhomocysteine or of S-adenosyl-3-thiopropylamine may be particularly valuable in this respect.     

Efficient Synthesis of Eriodictyol from l-Tyrosine in Escherichia coli

The health benefits of flavonoids for humans are increasingly attracting attention. Because the extraction of high-purity flavonoids from plants presents a major obstacle, interest has emerged in biosynthesizing them using microbial hosts. Eriodictyol is a flavonoid with anti-inflammatory and antioxidant activities. Its efficient synthesis has been hampered by two factors: the poor expression of cytochrome P450 and the low intracellular malonyl coenzyme A (malonyl-CoA) concentration in Escherichia coli. To address these issues, a truncated plant P450 flavonoid, flavonoid 3′-hydroxylase (tF3′H), was functionally expressed as a fusion protein with a truncated P450 reductase (tCPR) in E. coli. This allowed the engineered E. coli to produce eriodictyol from l-tyrosine by simultaneously coexpressing the fusion protein with tyrosine ammonia lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI). In addition, metabolic engineering was employed to enhance the availability of malonyl-CoA so as to achieve a new metabolic balance and rebalance the relative expression of genes to enhance eriodictyol accumulation. This approach made the production of eriodictyol 203% higher than that in the control strain. By using these strategies, the production of eriodictyol from l-tyrosine reached 107 mg/liter. The present work offers an approach to the efficient synthesis of other hydroxylated flavonoids from l-tyrosine or even glucose in E. coli.     

Purification and characterization of 1-aminocyclopropane-1-carboxylate synthase from apple fruits

1-Aminocyclopropane-1-carboxylate (ACC) synthase, a key enzyme in ethylene biosynthesis, was isolated and partially purified from apple (Malus sylvestris Mill.) fruits. Unlike ACC synthase isolated from other sources, apple ACC synthase is associated with the pellet fraction and can be solubilized in active form with Triton X-100. Following five purification steps, the solubilized enzyme was purified over 5000-fold to a specific activity of 100 micromoles per milligram protein per hour, and its purity was estimated to be 20 to 30%. Using this preparation, specific monoclonal antibodies were raised. Monoclonal antibodies against ACC synthase immunoglobulin were coupled to protein-A agarose to make an immunoaffinity column, which effectively purified the enzyme from a relatively crude enzyme preparation (100 units per milligram protein). As with the tomato enzyme, apple ACC synthase was inactivated and radiolabeled by its substrate S-adenosyl-l-methionine. Apple ACC synthase was identified to be a 48-kilodalton protein based on the observation that it was specifically bound to immunoaffinity column and it was specifically radiolabeled by its substrate S-adenosyl-l-methionine.     

Kinetic Mechanism of Protein N-terminal Methyltransferase 1

The protein N-terminal methyltransferase 1 (NTMT1) catalyzes the transfer of the methyl group from the S-adenosyl-l-methionine to the protein α-amine, resulting in formation of S-adenosyl-l-homocysteine and α-N-methylated proteins. NTMT1 is an interesting potential anticancer target because it is overexpressed in gastrointestinal cancers and plays an important role in cell mitosis. To gain insight into the biochemical mechanism of NTMT1, we have characterized the kinetic mechanism of recombinant NTMT1 using a fluorescence assay and mass spectrometry. The results of initial velocity, product, and dead-end inhibition studies indicate that methylation by NTMT1 proceeds via a random sequential Bi Bi mechanism. In addition, our processivity studies demonstrate that NTMT1 proceeds via a distributive mechanism for multiple methylations. Together, our studies provide new knowledge about the kinetic mechanism of NTMT1 and lay the foundation for the development of mechanism-based inhibitors.     

Concentrations of putrescine and polyamines and their enzymic synthesis during androgen-induced prostatic growth

1. Castration of adult rats resulted in marked decreases in the amounts of putrescine, spermidine and spermine in the ventral prostate gland. Spermidine concentrations decline rapidly over the first 11 days after androgen withdrawal, reaching a value of only 12% of normal controls. Spermine concentrations diminish more slowly, reaching 24% of normal within 11 days. The spermidine/spermine molar ratio falls from 0.9 to 0.46 under these conditions. Putrescine concentrations decrease by 70% at 7 days after castration and then remain constant for some days. 2. After daily injections of testosterone propionate to rats castrated 7 days previously, prostatic spermidine and putrescine concentrations increase significantly within 24h; normal or even greater values are observed within 8 and 4 days respectively. In contrast, the spermine concentration does not increase until 5 days after commencement of androgen treatment. 3. The activities of two enzymes involved in polyamine biosynthesis (ornithine decarboxylase and a putrescine-activated S-adenosyl-l-methionine decarboxylase system) were greatly decreased soon after castration: after 7 days the respective values were 15% of normal for ornithine decarboxylase and 7% of normal for putrescine-dependent decarboxylation of S-adenosyl-l-methionine. Injection of testosterone propionate into animals castrated 7 days previously induced a rapid increase in both enzymic activities: ornithine decarboxylase was doubled in 6h, and increased three- to four-fold within 48h, whereas the putrescine-dependent decarboxylation of S-adenosyl-l-methionine doubled in 3h and increased tenfold within 48h of commencement of daily androgen treatments. 4. The activity of these enzyme systems was very low in the ventral prostates of hypophysectomized rats and was increased by administration of testosterone in a manner similar to that found in castrated rats. 5. Alterations in the activity of two ventral-prostate enzymes involved in ornithine production (arginase) and utilization (ornithine–2-oxoglutarate transaminase) that result from changes in the androgenic status of rats are described. 6. The findings presented suggest that the activities of ornithine decarboxylase and the putrescine-dependent S-adenosyl-l-methionine decarboxylase system, rather than ornithine concentrations, are rate-limiting for the formation of putrescine and polyamines in rat ventral prostate. 7. The relation of polyamines to androgen-induced prostatic growth is discussed with particular reference to the biosynthesis of proteins and nucleic acids.     

Crystal structure of the heme d1 biosynthesis enzyme NirE in complex with its substrate reveals new insights into the catalytic mechanism of S-adenosyl-L-methionine-dependent uroporphyrinogen III methyltransferases

During the biosynthesis of heme d₁, the essential cofactor of cytochrome cd₁ nitrite reductase, the NirE protein catalyzes the methylation of uroporphyrinogen III to precorrin-2 using S-adenosyl-l-methionine (SAM) as the methyl group donor. The crystal structure of Pseudomonas aeruginosa NirE in complex with its substrate uroporphyrinogen III and the reaction by-product S-adenosyl-l-homocysteine (SAH) was solved to 2.0 Å resolution. This represents the first enzyme-substrate complex structure for a SAM-dependent uroporphyrinogen III methyltransferase. The large substrate binds on top of the SAH in a “puckered” conformation in which the two pyrrole rings facing each other point into the same direction either upward or downward. Three arginine residues, a histidine, and a methionine are involved in the coordination of uroporphyrinogen III. Through site-directed mutagenesis of the nirE gene and biochemical characterization of the corresponding NirE variants the amino acid residues Arg-111, Glu-114, and Arg-149 were identified to be involved in NirE catalysis. Based on our structural and biochemical findings, we propose a potential catalytic mechanism for NirE in which the methyl transfer reaction is initiated by an arginine catalyzed proton abstraction from the C-20 position of the substrate.     

In Vivo Biotinylation of Bacterial Magnetic Particles by a Truncated Form of Escherichia coli Biotin Ligase and Biotin Acceptor Peptide

Escherichia coli biotin ligase can attach biotin molecules to a lysine residue of biotin acceptor peptide (BAP), and biotinylation of particular BAP-fused proteins in cells was carried out by coexpression of E. coli biotin ligase (in vivo biotinylation). This in vivo biotinylation technology has been applied for protein purification, analysis of protein localization, and protein-protein interaction in eukaryotic cells, while such studies have not been reported in bacterial cells. In this study, in vivo biotinylation of bacterial magnetic particles (BacMPs) synthesized by Magnetospirillum magneticum AMB-1 was attempted by heterologous expression of E. coli biotin ligase. To biotinylate BacMPs in vivo, BAP was fused to a BacMP surface protein, Mms13, and E. coli biotin ligase was simultaneously expressed in the truncated form lacking the DNA-binding domain. This truncation-based approach permitted the growth of AMB-1 transformants when biotin ligase was heterologously expressed. In vivo biotinylation of BAP on BacMPs was confirmed using an alkaline phosphatase-conjugated antibiotin antibody. The biotinylated BAP-displaying BacMPs were then exposed to streptavidin by simple mixing. The streptavidin-binding capacity of BacMPs biotinylated in vivo was 35-fold greater than that of BacMPs biotinylated in vitro, where BAP-displaying BacMPs purified from bacterial cells were biotinylated by being mixed with E. coli biotin ligase. This study describes not only a simple method to produce biotinylated nanomagnetic particles but also a possible expansion of in vivo biotinylation technology for bacterial investigation.Biotin/streptavidin binding is the strongest noncovalent interaction known in nature (K_d [dissociation constant], ∼10⁻¹⁵ M) (10), and this tight binding is one of the most general tools for biological research and has been widely used for biomolecular detection (11, 12), immobilization (14, 19), and recovery (15). Therefore, it is of great significance to biotinylate biomolecules, in particular, proteins without functional inhibition. For this purpose, the method for site-selective biotinylation of proteins had been developed using biotin ligase. Biotin ligase catalyzes the posttranslational biotinylation of biotin enzymes, such as acetyl coenzyme A (acetyl-CoA) carboxylase, and introduces biotin into a specific lysine residue of a biotin carboxyl carrier protein (BCCP), a subunit of biotin enzymes (13). In early studies, BCCP (∼100 amino acid residues) had been fused with the proteins of interest for biotinylation by biotin ligase (7); however, there was a concern that fused BCCP might disrupt the function of target proteins. Recently, biotin acceptor peptides (BAPs) had replaced BCCP due to the advantage of small size. BAPs, with 15 to 23 amino acid residues, were screened from a peptide library as peptide tags biotinylated by Escherichia coli biotin ligase (4, 25). BAP-fused proteins can be biotinylated outside the cells by adding biotin and purified E. coli biotin ligase with Mg²⁺ and ATP (in vitro biotinylation). Furthermore, it is also possible to biotinylate BAP-fused proteins inside the cells with coexpression of E. coli biotin ligase (in vivo biotinylation) because BAP is specifically recognized only by E. coli biotin ligase. This in vivo biotinylation technology has been applied in eukaryotic cells to purify the proteins by using streptavidin-immobilized resin (8, 24, 28), because biotin/streptavidin interaction permits stringent washing to eliminate the nonspecific binding. Specific biotinylation can be applied also for protein localization analysis. Using fluorophore- or gold nanoparticle-labeled streptavidin, biotinylated proteins were clearly observed in a previous study (27). Recently, a novel technique to detect protein-protein interaction by fusing BAP and biotin ligase was developed by Ting''s group. BAP and biotin ligase were fused to different two proteins, and then the interaction of these proteins was successfully evaluated via biotinylation of BAP (9). In vivo biotinylation technology using heterologously expressed E. coli biotin ligase should be equally useful for prokaryotes; however, such studies have not been reported for bacterial cells.Magnetospirillum magneticum AMB-1, a magnetotactic bacterium, synthesizes intracellular nanosized bacterial magnetic particles (BacMPs) of 50 to 100 nm; these are surrounded by a lipid bilayer membrane, possess a single magnetic domain of magnetite, and exhibit strong ferrimagnetism (18). Furthermore, functional proteins have been displayed on BacMP surfaces through gene fusion techniques (21, 30, 31). BacMP membrane proteins, including Mms13, were used as anchor proteins; this approach permits functional proteins to be localized efficiently and oriented appropriately on BacMPs (31). We recently reported a novel method for the simple production of biotin-labeled magnetic particles through protein display techniques, where introduction of the biotin moiety onto BacMPs was carried out by the endogenous biotin ligase (17). For the biotinylation of BacMPs, we screened the gene encoding BCCP in the AMB-1 genome and displayed it on the surface of BacMPs using an anchor protein, Mms13. BCCP-displaying BacMPs were biotinylated by endogenous AMB-1 biotin ligase in the cells with high efficiency. This in vivo modification approach could be applied for construction of BacMP-quantum dot nanocomposites toward multicolor labeling of cancer cells, where BCCP and antibody carrier protein (protein G) were simultaneously displayed in tandem (16). However, the size of BCCP, with 149 amino acid residues and a mass of 15.6 kDa, makes it rather large for use as a labeling tag. Although it would be preferable to use a smaller peptide, BAP, for the tag to minimize effects on the flanking proteins for future applications, BAP was not recognized and biotinylated by endogenous AMB-1 biotin ligase (17).In this study, in vivo biotinylation of BacMPs was attempted by heterologous expression of E. coli biotin ligase and Mms13-BAP fusion protein in AMB-1 cells. First, the method for effective expression of E. coli biotin ligase in bacterial cells was optimized. Then site-selective biotinylation of BAP on BacMPs was confirmed. Finally, the obvious advantage of in vivo biotinylation of BAP-displaying BacMPs compared with the in vitro biotinylation method was demonstrated.     

Turnover of 1-aminocyclopropane-1-carboxylic Acid synthase protein in wounded tomato fruit tissue

Ethylene production in plant tissues declines rapidly following induction, and this decline is due to a rapid decrease in the activity of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, a key enzyme in ethylene biosynthesis. To study the nature of the rapid turnover of ACC synthase in vivo, proteins in wounded ripening tomato (Lycopersicon esculentum) fruit discs were radiolabeled with [³⁵S]methionine, followed by a chase with nonradioactive methionine. Periodically, the radioactive ACC synthase was isolated with an immunoaffinity gel and analyzed. ACC synthase protein decayed rapidly in vivo with an apparent half-life of about 58 min. This value for protein turnover in vivo is similar to that previously reported for activity half-life in vivo and substrate-dependent enzyme inactivation in vitro. Carbonylcyanide-m-chlorophenylhydrazone and 2,4-dinitrophenol, potent uncouplers of oxidative phosphorylation, strongly inhibited the rapid decay of ACC synthase protein in the tissue. Degradation of this enzyme protein was moderately inhibited by the administration of aminooxyacetic acid, a competitive inhibitor of ACC synthase with respect to its substrate S-adenosyl-l-methionine, α,α′-dipyridyl, and phenylmethanesulfonyl fluoride or leupeptin, serine protease inhibitors. These results support the notion that the substrate S-adenosyl-l-methionine participates in the rapid inactivation of the enzyme in vivo and suggest that some ATP-dependent processes, such as the ubiquitin-requiring pathway, are involved in the degradation of ACC synthase proteins.     

Terpenoid Metabolism in Plastids : Localization of alpha-Tocopherol Synthesis in Capsicum Chromoplasts

The synthesis of α-tocopherol from 2,3-dimethylphytylquinol and S-adenosyl-l-methionine was achieved using Capsicum annuum fruit chromoplasts. The enzymes involved in the cyclization (2,3-dimethyl-phytylquinol cyclase) and methylation (S-adenosyl methionine:γ-tocopherol methyl-transferase) are both localized in the chromoplast membrane fraction (envelopes and/or a-chlorophyll lamellae), in contrast to the stroma fraction.     

Biotin biosynthetic pathway in recombinant strains of Escherichia coli overexpressing bio genes: evidence for a limiting step upstream from KAPA

The effect of the overexpression of the bioABFCD operon on the biotin biosynthetic pathway was investigated in an Escherichia coli K12 bioR mutant with a chromosomal deletion for the biotin operon. When transformed with a multicopy number plasmid containing bioABFCD, this strain synthetized 10,000 times more biotin than a wild-type E. coli strain. In order to further increase biotin production, the bioA and bioB operons were subcloned into plasmids with stronger promoters and in some cases optimal ribosome binding sites. The new constructions led to the accumulation of large amounts of soluble Bio proteins (although not BioC) but did not improve biotin production. In all the constructed strains, BioA, BioD, and BioB activities were greatly amplified but these activitie did not correlate with the level of protein syntthesis. These strains accumulated only low levels of vitamers, auggesting that the major limiting step for higher biotin production occurs upstream from the first intermediate of the Bio pathway we assayed (7,keto-8-aminopelargonic acid). As BioC overproduction was shown to impair cell growth, we could not determine if this early step of pathway was limiting. Correspondence to: S. Lévy-Schil     

Chemical and biological properties of an extracellular lipopolysaccharide from Escherichia coli grown under lysine-limiting conditions

Lipopolysaccharide was prepared from the extracellular lipoglycopeptide produced by the lysine-requiring mutant Escherichia coli A.T.C.C. 12408 grown under lysine-limiting conditions. The lipid moiety, containing glucosamine phosphate and four fatty acids (lauric acid, myristic acid, β-hydroxymyristic acid and palmitic acid) corresponded in composition to lipid A of known bacterial lipopolysaccharides. The components of the polysaccharide moiety were d-glucose, d-galactose, l-glycero-d-manno-heptose, 3-deoxy-2-oxo-octonic acid, ethanolamine and phosphate. These are the constituents of the polysaccharide of the cell-wall antigens from rough strains of E. coli. Lipopolysaccharides were also prepared from whole cells of E. coli 12408 grown with excess or limited amounts of lysine; they were identical in carbohydrate composition with the extracellular lipopolysaccharide. The biological properties of this material also resembled those of known lipopolysaccharides; it was antigenic, pyrogenic, toxic and had adjuvant activity.     

Purification and Characterization of S-Adenosyl-l-methionine:6a-Hydroxymaackiain 3-O-Methyltransferase from Pisum sativum

The isoflavonoid phytoalexin pisatin is synthesized by Pisum sativum in response to microbial infection and certain other forms of stress. An enzyme which synthesizes pisatin by methylating the 3-hydroxyl of (+)6a-hydroxymaackiain (HMK) was extracted from CuCl₂-stressed pea seedlings. The enzyme was enriched 370-fold by (NH₄)₂SO₄ precipitation, DEAE chromatography, chromatofocusing, and hydrophobic interaction chromatography (HIC), to a specific activity of 8.2 microkatals per gram protein. Enzyme activity profiles from chromatofocusing and HIC columns suggested the presence of two isozymes, of pl 5.2 and 4.9. Nondenaturing gel filtration of the HIC-purified enzyme gave a single peak of activity at the same elution volume as BSA (66 kilodaltons); the active fractions showed two proteins upon SDS-PAGE, of M_r 66,000 and 43,000. The smaller protein was most abundant in chromatographic fractions containing peak enzyme activity throughout purification. In a partially purified preparation, this 43 kilodalton protein was the only one photoaffinity labelled by [³H]S-adenosyl-l-methionine. The purified enzyme preferred the (+) over the (−) stereoisomer of HMK and other pterocarpans; overall, (+)HMK was the best substrate. K_m values were 2.3 micromolar for (+)HMK and 35 micromolar for S-adenosyl-l-methionine. The methyltransferase had a pH optimum of 7.9 and no apparent divalent cation requirement.     

One-Pot Production of l-threo-3-Hydroxyaspartic Acid Using Asparaginase-Deficient Escherichia coli Expressing Asparagine Hydroxylase of Streptomyces coelicolor A3(2)

We developed a novel process for efficient synthesis of l-threo-3-hydroxyaspartic acid (l-THA) using microbial hydroxylase and hydrolase. A well-characterized mutant of asparagine hydroxylase (AsnO-D241N) and its homologous enzyme (SCO2693-D246N) were adaptable to the direct hydroxylation of l-aspartic acid; however, the yields were strictly low. Therefore, the highly stable and efficient wild-type asparagine hydroxylases AsnO and SCO2693 were employed to synthesize l-THA. By using these recombinant enzymes, l-THA was obtained by l-asparagine hydroxylation by AsnO followed by amide hydrolysis by asparaginase via 3-hydroxyasparagine. Subsequently, the two-step reaction was adapted to one-pot bioconversion in a test tube. l-THA was obtained in a small amount with a molar yield of 0.076% by using intact Escherichia coli expressing the asnO gene, and thus, two asparaginase-deficient mutants of E. coli were investigated. A remarkably increased l-THA yield of 8.2% was obtained with the asparaginase I-deficient mutant. When the expression level of the asnO gene was enhanced by using the T7 promoter in E. coli instead of the lac promoter, the l-THA yield was significantly increased to 92%. By using a combination of the E. coli asparaginase I-deficient mutant and the T7 expression system, a whole-cell reaction in a jar fermentor was conducted, and consequently, l-THA was successfully obtained from l-asparagine with a maximum yield of 96% in less time than with test tube-scale production. These results indicate that asparagine hydroxylation followed by hydrolysis would be applicable to the efficient production of l-THA.     

Inactivation of 1-Aminocyclopropane-1-Carboxylate Synthase by l-Vinylglycine as Related to the Mechanism-Based Inactivation of the Enzyme by S-Adenosyl-l-Methionine

The pyridoxal phosphate-dependent 1-aminocyclopropane-1-carboxylate (ACC) synthase catalyzes the conversion of S-adenosyl-l-methionine (AdoMet) to ACC, and is inactivated by AdoMet during the reaction. l-Vinylglycine was found to be a competitive inhibitor of the enzyme, and to cause a time-dependent inactivation of the enzyme. The inactivation required the presence of pyridoxal phosphate and followed pseudo-first-order kinetics at various concentrations of l-vinylglycine. The Michaelis constant for l-vinylglycine in the inactivation reaction (K_inact) was 3.3 millimolar and the maximum rate constant (k_max) was 0.1 per minute. These findings, coupled with the previous observations that the suicidal action of AdoMet involved a covalent linkage of the aminobutyrate portion of AdoMet to the enzyme, support the view that the mechanism-based inactivation of ACC synthase by the substrate AdoMet proceeds through the formation of a vinylglycine-ACC synthase complex as an intermediate.     

Functional replacement of the endogenous tyrosyl-tRNA synthetase–tRNATyr pair by the archaeal tyrosine pair in Escherichia coli for genetic code expansion

Non-natural amino acids have been genetically encoded in living cells, using aminoacyl-tRNA synthetase–tRNA pairs orthogonal to the host translation system. In the present study, we engineered Escherichia coli cells with a translation system orthogonal to the E. coli tyrosyl-tRNA synthetase (TyrRS)–tRNA^Tyr pair, to use E. coli TyrRS variants for non-natural amino acids in the cells without interfering with tyrosine incorporation. We showed that the E. coli TyrRS–tRNA^Tyr pair can be functionally replaced by the Methanocaldococcus jannaschii and Saccharomyces cerevisiae tyrosine pairs, which do not cross-react with E. coli TyrRS or tRNA^Tyr. The endogenous TyrRS and tRNA^Tyr genes were then removed from the chromosome of the E. coli cells expressing the archaeal TyrRS–tRNA^Tyr pair. In this engineered strain, 3-iodo-l-tyrosine and 3-azido-l-tyrosine were each successfully encoded with the amber codon, using the E. coli amber suppressor tRNATyr and a TyrRS variant, which was previously developed for 3-iodo-l-tyrosine and was also found to recognize 3-azido-l-tyrosine. The structural basis for the 3-azido-l-tyrosine recognition was revealed by X-ray crystallography. The present engineering allows E. coli TyrRS variants for non-natural amino acids to be developed in E. coli, for use in both eukaryotic and bacterial cells for genetic code expansion.     

Reassignment of a rare sense codon to a non-canonical amino acid in Escherichia coli

The immutability of the genetic code has been challenged with the successful reassignment of the UAG stop codon to non-natural amino acids in Escherichia coli. In the present study, we demonstrated the in vivo reassignment of the AGG sense codon from arginine to l-homoarginine. As the first step, we engineered a novel variant of the archaeal pyrrolysyl-tRNA synthetase (PylRS) able to recognize l-homoarginine and l-N⁶-(1-iminoethyl)lysine (l-NIL). When this PylRS variant or HarRS was expressed in E. coli, together with the AGG-reading tRNA^Pyl_CCU molecule, these arginine analogs were efficiently incorporated into proteins in response to AGG. Next, some or all of the AGG codons in the essential genes were eliminated by their synonymous replacements with other arginine codons, whereas the majority of the AGG codons remained in the genome. The bacterial host''s ability to translate AGG into arginine was then restricted in a temperature-dependent manner. The temperature sensitivity caused by this restriction was rescued by the translation of AGG to l-homoarginine or l-NIL. The assignment of AGG to l-homoarginine in the cells was confirmed by mass spectrometric analyses. The results showed the feasibility of breaking the degeneracy of sense codons to enhance the amino-acid diversity in the genetic code.     

Small Multidrug Resistance Protein EmrE Reduces Host pH and Osmotic Tolerance to Metabolic Quaternary Cation Osmoprotectants

The small multidrug resistance (SMR) transporter protein EmrE in Escherichia coli is known to confer resistance to toxic antiseptics classified as quaternary cation compounds (QCCs). Naturally derived QCCs synthesized during metabolic activities often act as osmoprotectants, such as betaine and choline, and participate in osmotic homoestasis. The goal of this study was to determine if EmrE proteins transport biological QCC-based osmoprotectants. Plasmid-encoded copies of E. coli emrE and the inactive variant emrE-E14C (emrE with the E→C change at position 14) were expressed in various E. coli strains grown in either rich or minimal media at various pHs (5 to 9) and under hypersaline (0.5 to 1.0 M NaCl and KCl) conditions to identify changes in growth phenotypes induced by osmoprotectant transport. The results demonstrated that emrE expression reduced pH tolerance of E. coli strains at or above neutral pH and when grown in hypersaline media at or above NaCl or KCl concentrations of 0.75 M. Hypersaline growth conditions were used to screen QCC osmoprotectants betaine, choline, l-carnitine, l-lysine, l-proline, and l-arginine. The study identified that betaine and choline are natural QCC substrates of EmrE.