The whole-cell immobilization of D-hydantoinase-engineered Escherichia coli for D-CpHPG biosynthesis

Background: D-Hydroxyphenylglycine is considered to be an important chiral molecular building-block of antibiotic reagents such as pesticides, and β-lactam antibiotics. The process of its production is catalyzed by D-hydantoinase and D-carbamoylase in a two-step enzyme reaction. How to enhance the catalytic potential of the two enzymes is valuable for industrial application. In this investigation, an Escherichia coli strain genetically engineered with D-hydantoinase was immobilized by calcium alginate with certain adjuncts to evaluate the optimal condition for the biosynthesis of D-carbamoyl-p-hydroxyphenylglycine (D-CpHPG), the compound further be converted to D-hydroxyphenylglycine (D-HPG) by carbamoylase. Results: The optimal medium to produce D-CpHPG by whole-cell immobilization was a modified Luria-Bertani (LB) added with 3.0% (W/V) alginate, 1.5% (W/V) diatomite, 0.05% (W/V) CaCl2 and 1.00 mM MnCl2.The optimized diameter of immobilized beads for the whole-cell biosynthesis here was 2.60 mm. The maximized production rates of D-CpHPG were up to 76%, and the immobilized beads could be reused for 12 batches. Conclusions: This investigation not only provides an effective procedure for biological production of D-CpHPG, but gives an insight into the whole-cell immobilization technology.

Metabolic basis for the self-referential genetic code.

An investigation of the biosynthesis pathways producing glycine and serine was necessary to clarify an apparent inconsistency between the self-referential model (SRM) for the formation of the genetic code and the model of coevolution of encodings and of amino acid biosynthesis routes. According to the SRM proposal, glycine was the first amino acid encoded, followed by serine. The coevolution model does not state precisely which the first encodings were, only presenting a list of about ten early assignments including the derivation of glycine from serine-this being derived from the glycolysis intermediate glycerate, which reverses the order proposed by the self-referential model. Our search identified the glycine-serine pathway of syntheses based on one-carbon sources, involving activities of the glycine decarboxylase complex and its associated serine hydroxymethyltransferase, which is consistent with the order proposed by the self-referential model and supports its rationale for the origin of the genetic code: protein synthesis was developed inside an early metabolic system, serving the function of a sink of amino acids; the first peptides were glycine-rich and fit for the function of building the early ribonucleoproteins; glycine consumption in proteins drove the fixation of the glycine-serine pathway.

Palythine-threonine, a major novel mycosporine-like amino acid (MAA) isolated from the hermatypic coral Pocillopora capitata.

Using a high-resolution reverse-phase liquid chromatography method we found that the tissues of the hermatypic coral Pocillopora capitata (collected in Santiago Bay, Mexico) contain a high diversity of primary and secondary mycosporine-like amino acids (MAAs) typical of some reef-building coral species: mycosporine-glycine, shinorine, porphyra-334, mycosporine-methylamine-serine, mycosporine-methylamine-threonine, palythine-serine, palythine and one additional novel predominant MAA, with an absorbance maximum of 320 nm. Here we document the isolation and characterization of this novel MAA from the coral P. capitata. Using low multi-stage mass analyses of deuterated and non deuterated compounds, high-resolution mass analyses (Time of Flight, TOF) and other techniques, this novel compound was characterized as palythine-threonine. Palythine-threonine was also present in high concentrations in the corals Pocillopora eydouxi and Stylophora pistillata indicating a wider distribution of this MAA among reef-building corals. From structural considerations we suggest that palythine-threonine is formed by decarboxylation of porphyra-334 followed by demethylation of mycosporine-methylamine-threonine.

Glycine is not formed through the amino transferase reaction in human or rat placenta.

The fetus has a substantial demand for glycine, which is satisfied in part by placental formation. The ability to form glycine through the activity of alanine:glyoxylate aminotransferase enzyme was measured in placentae from normal term human pregnancies and placentae from rats at day 20 of gestation. There was no detectable enzyme activity in either human or rat placentae, although activity was measured in rat liver. It is concluded that in the placenta glycine is only formed from serine through the activity of serine hydroxymethyl transferase enzyme, which uses folate as a cofactor, because there are no other known metabolic pathways for endogenous glycine production.

Glycine is not formed through the amino transferase reaction in human or rat placenta

The fetus has a substantial demand for glycine, which is satisfied in part by placental formation. The ability to form glycine through the activity of alanine: gloxylate aminotransferase enzyme was measured in placentae from normal term human pregnancies and placentae from rats at day 20 of gestation. There was no detectable enzyme activity in either human or rat placentae, although activity was measured in rat liver. It is concluded that in the placenta glycine is only formed from serine through the activity of serine hydroxymethyl transferase enzyme, which uses folate as a cofactor, because there is no other known metabolic pathways for endogenous glycine production.(AU)

Urinary 5-L-oxoproline (pyroglutamic acid) excretion is greater in infants in Jamaica than in infants in England.

OBJECTIVE: To determine the pattern of excretion in urine of 5-L-oxoproline, as a measure of glycine status, during the first six weeks of life in Jamaican infants. DESIGN: Spot samples of urine were collected from term and preterm infants at birth and longitudinally to four weeks of age, or at six weeks of age. 5-L-oxoproline was isolated by column chromatography and hydrolysed to L-glutamic acid, which was measured enzymatically and the results expressed relative to creatinine excretion. SETTING: Maternity wards and postnatal clinic of the University Hospital of the West Indies. SUBJECTS: African-Caribbean infants, 19 term and 21 preterm, from birth to four weeks of age, and 79 term infants at six weeks of age. RESULTS: There were no differences between term and preterm infants. Excretion of 5-L-oxoproline increased progressively from birth, 141 mumol/mmol creatinine, to 270 mumol/mmol creatinine at four weeks of age. At six weeks of age, excretion was significantly greater than at birth or four weeks of age, 525 mumol/mmol creatinine. Compared with infants born in England, the excretion of 5-L-oxoproline was not different at birth, but was significantly greater in Jamaican infants at six weeks of age. CONCLUSIONS: Glycine status, indicated by increased excretion of 5-L-oxoproline, is marginal in Jamaican infants at six weeks of age, and this possibly reflects a limitation in the endogenous biosynthesis of glycine due to a dietary limitation of folate or vitamin B-12.

Urinary 5-L-oxoproline (pyroglutamic acid) excretion is greater in infants in Jamaican than in infants in England

OBJECTIVE: To determine the pattern of excretion in urine of 5-L-oxoproline, as a measure of glycine status, during the first six weeks of life in Jamaican infants. DESIGN: Spot samples of urine were collected from term and preterm infants at birth and longitudinally to four weeks of age, or at six weeks of age. 5-L-oxoproline was isolated by column chromatography and hydrolysed to L-glutamic acid, which was measured enzymatically and results expressed relative to creatinine excretion. SETTING: Maternity wards and postnatal clinic of the University Hospital of the West Indies. SUBJECTS: African-Caribbean infants, 19 term and 21 preterm, from birth to four weeks of age, and 79 term infants at six weeks of age. RESULTS: There was no difference between term and preterm infants. Excretion of 5-L-oxoproline increased progressively from birth, 141 æmol/mmol creatinine at four weeks of age. At six weeks of age, excretion was significantly greater than at birth or four weeks of age, 525 æmol/mmol creatinine. Compared with infant born in England, the excretion of 5-L-oxoproline was not different at birth, but was significantly greater in Jamaican infant at six weeks of age. CONCLUSIONS: Glycine status, indicated by increased excretion of 5-L-oxoproline, is marginal in Jamaican infants at six weeks of age, and this is possibly reflects a limitation in the endogenous biosynthesis of glycine due to a dietary limitation of folate or vitamin B-12. (AU)

Evidence for neuronal release of isotopically labelled glycine from the rat ventral medullary surface in vivo

Spontaneous and stimulus-induced release of isotopically labelled glycine was studied in the superfused rat dorsal or ventral medullary surface in vivo. Superfusion of the ventral medullary surface of anesthetized (urethane, 1.2 g/kg, ip) male adult Wistar rats (250-350 g) with high K+ (40 mM) surrogate cerebrospinal fluid (CSF) produced an average increase of 45 per cent over the spontaneous efflux of exogenously applied glycine (N = 5, P<0.01). In experiments in which the calcium of the CSF was replaced by an equimolar amount of magnesium, the increase in glycine efflux in response to high K+ was reduced to 15 per cent, a value not statistically different from that observed in control experiments (N = 6). Veratridine stimulation evoked a large (80 per cent) increase in glycine efflux (N = 5, P<0.001), which was inhibited by tetrodotoxin. High potassium or veratridine failed to modify spontaneous release of glycine on the dorsal medullary surface. Results obtained in control experiments showed that neither high K+ nor veratridine is effective in modifying spontaneous efflux of [(3)H]-leucine or [(3)H]-inulin on the ventral or dorsal medullary surface. These data support the hypothesis that glycine is a neurotransmitter on the ventral medullary surface and that it may be part of neural pathways involved in cardiorespiratory regulation present in this region.

Inhibición de la glicosilación no enzimática de la hemoglobina en la diabetes mellitus / Inhibition of non-enzymatic glycosilation of hemoglobin in diabetes mellitus

Se estudió el efecto inhibidor del aminoácido glicina sobre la glicosilación no enzimática de la hemoglobina en la diabetes experimental de ratas Wistar con estreptozotocina. La hemoglobina glicosilada de las ratas diabéticas fue de 4.2 ñ 0.38 por ciento y la de las diabéticas que tomaron glicina al 1 por ciento en el agua de bebida ad libitum fue de 2.90 ñ 0.37 por ciento (p = 0.00005). Un grupo de 30 personas diabéticas tipo II y 8 de tipo I tomaron glicina disuelta en agua: 20 gramos diarios (4 tomas de 5 g cada 6 horas) durante tiempos variables: de 3 hasta 56 meses. La hemoglobina glicosilada promedio de los diabéticos antes de tomar la clicina fue de 12.8 ñ 3.3 por ciento y después fue de 8.3 ñ 2.2 por ciento con un valor de p= 7 x 10-12 (prueba de rangos señalados de Wilcoxon)

Limitations of 3-phenylpropionylglycine in early screening for medium-chain acyl-coenzyme A dehydrogenase deficiency.

Screening for medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency by urinary 3-phenylpropionylglycine may not be reliable in early infancy because young infants are not colonized with adult-type colonic flora. In this study we delineated the microbes that produce 3-phenylpropionic acid, the precursor of 3-phenylpropionylglycine. We found that the use of some antibiotics may alter gut flora, thereby confounding this method of screening for MCAD deficiency.