Molecular engineering and immobilization of lysine decarboxylase for synthesis of 1, 5-diaminopentane: a review / 生物工程学报

1, 5-diaminopentane, also known as cadaverine, is an important raw material for the production of biopolyamide. It can be polymerized with dicarboxylic acid to produce biopolyamide PA5X whose performances are comparable to that of the petroleum-based polyamide materials. Notably, biopolyamide uses renewable resources such as starch, cellulose and vegetable oil as substrate. The production process does not cause pollution to the environment, which is in line with the green and sustainable development strategy. The biosynthesis of 1, 5-diaminopentane mainly includes two methods: the de novo microbial synthesis and the whole cell catalysis. Lysine decarboxylase as the key enzyme for 1, 5-diaminopentane production, mainly includes an inducible lysine decarboxylase CadA and a constituent lysine decarboxylase LdcC. Lysine decarboxylase is a folded type Ⅰ pyridoxal-5' phosphate (PLP) dependent enzyme, which displays low activity and unstable structure, and is susceptible to deactivation by environmental factors in practical applications. Therefore, improving the catalytic activity and stability of lysine decarboxylase has become a research focus in this field, and molecular engineering and immobilization are the mainly approaches. Here, the mechanism, molecular engineering and immobilization strategies of lysine decarboxylase were reviewed, and the further strategies for improving its activity and stability were also prospected, with the aim to achieve efficient production of 1, 5-diaminopentane.

Micronutrients: Speculation on Inborn Errors, Nutrigenomics, Evolution, the Microbiome, and Nutritional Immunity

Abstract Many micronutrients or cofactors derived from micronutrients are highly reactive, hence their role in catalysis of reactions by enzymes. The concentration of cofactors has to be kept low to avoid unwanted reactions while allowing them to bind to the (apo)enzymes that need them. A new disorder causing B6-responsive epilepsy (proline synthetase cotranscribed bacterial homologue deficiency) is probably due to the absence of an important intracellular pyridoxal phosphate chaperone. The availability of some micronutrients varies by orders of magnitude in different geographical areas. Selenium is both essential and toxic, and during evolution, different populations have had to adapt to this differing availability. An "inborn error of metabolism (IEM)" in a low selenium area of China may be a selective advantage in a high selenium area and vice versa; the concept of nutrigenomics is an important one for micronutrients. The gut flora may make an important contribution to vitamin synthesis. This is difficult to study, but experiments can be undertaken with the nematode, Caenorhabditis elegans (with or without an IEM) and a single clone of Escherichia coli (with or without an IEM) as food and gut flora. This model shows that the gut microbiome can have profound influences on the folate cycle and associated vitamins. Our innate immune system makes use of the micronutrient requirements of pathogens and can deprive a pathogen of essential micronutrient(s) or expose it to toxic levels. It is not surprising, therefore, that some mutations affecting the way the host handles micronutrients can confer an advantage in resistance to infection and this may have acted as a selective advantage during evolution. This will be discussed by reference to the relationship of inborn errors to resistance to malaria. Conversely, other inborn errors of micronutrient metabolism are likely to make it more difficult for the host to use nutritional immunity to fight infection; this probably accounts for the list of infections that are more serious in patients with hereditary haemochromatosis.

Clinical characteristics and genetic analysis of 2 children with pyridox (am)ine-5'-phosphate oxidase deficiency / 中华实用儿科临床杂志

Objective To analyze the clinical characteristics and pyridox (am)ine-5'-phosphate oxidase(PNPO) gene mutations in 2 patients with PNPO deficiency.Methods The identical twin brothers were diagnosed at the Department of Pediatrics of Peking University First Hospital in February 2016.The clinical presentations,course of treatment,blood biochemistry,metabolic screening,EEG,brain magnetic resonance imaging (MRI) and epilepsy-related genes detection (including PNPO gene) of them were analyzed.Results These 2 patients were born at 35 +5weeks gestation and had asphyxia after birth.The seizures started within the first day,which was uncontrolled by various antiepileptic drugs.EEG showed atypical hypsarrhythmia or multifocal epileptiform discharges.MRI showed nonspecific abnormality.Pyridoxine was used as monotherapy or combination with various antiepileptic drugs during the treatment.Seizures had ever been controlled by pyridoxine alone for up to 1 month.Antiepileptic drugs were withdrawn gradually under the circumstances of seizures persisting when they became 5 years old.During the past year,pyridoxine was used alone.They still had seizures at their age of 6 years and 4 months.Blood metabolic screening showed that the level of arginine,asparaginic acid and methionine decreased.Urinary metabolic screening showed vanillic acid elevating prominently in both patients,49.78 and 36.60 times beyond normal,respectively.Genetic analysis showed compound heterozygous variants ofPNPO gene in both patients:c.445_448del (p.P150RfsX27) and c.481C ＞T (p.R161C).These 2variants were not reported previously.After definite diagnosis was made,pyridoxine was replaced by pyridoxal-5'-phosphate (PLP) immediately.Seizures increased slightly at the initial treating with PLP,then reduced gradually and were controlled eventually.Psychomotor development was severely delayed in 2 patients.Conclusions Infantile onset intractable seizures in these 2 patients responded to pyridoxine.The results of blood and urinary metabolic screening suggest the possibility of PNPO deficiency.This is the first time to report patients with PNPO deficiency diagnosed by PNPO gene mutations in China.Seizures could be controlled by PLP monotherapy eventually.

Evaluation of vitamin B6 intake and status of 20- to 64-year-old Koreans

BACKGROUND/OBJECTIVES: Recent research regarding vitamin B6 status including biochemical index is limited. Thus, this study estimated intakes and major food sources of vitamin B6; determined plasma pyridoxal 5'-phosphate (PLP); and assessed vitamin B6 status of Korean adults. MATERIALS/METHODS: Three consecutive 24-h diet recalls and fasting blood samples were collected from healthy 20- to 64-year-old adults (n = 254) living in the Seoul metropolitan area, cities of Kwangju and Gumi, Korea. Vitamin B6 intake and plasma PLP were analyzed by gender and by vitamin B6 supplementation. Pearson's correlation coefficient was used to determine associations of vitamin B6 intake and plasma PLP. RESULTS: The mean dietary and total (dietary plus supplemental) vitamin B6 intake was 1.94 +/- 0.64 and 2.41 +/- 1.45 mg/day, respectively. Median (50th percentile) dietary intake of men and women was 2.062 and 1.706 mg/day. Foods from plant sources provided 70.61% of dietary vitamin B6 intake. Only 6.3% of subjects consumed total vitamin B6 less than Estimated Average Requirements. Plasma PLP concentration of all subjects was 40.03 +/- 23.71 nmol/L. The concentration of users of vitamin B6 supplements was significantly higher than that of nonusers (P < 0.001). Approximately 16% of Korean adults had PLP levels < 20 nmol/L, indicating a biochemical deficiency of vitamin B6, while 19.7% had marginal vitamin B6 status. Plasma PLP concentration showed positive correlation with total vitamin B6 intake (r = 0.40984, P < 0.0001). CONCLUSIONS: In this study, vitamin B6 intake of Korean adults was generally adequate. However, one-third of subjects had vitamin B6 deficiency or marginal status. Therefore, in some adults in Korea, consumption of vitamin B6-rich food sources should be encouraged.

Theoretical studies on the pyridoxal-5'-phosphate dependent enzyme dopa decarboxylase: Effect of Thr 246 residue on the co-factor-enzyme binding and reaction mechanism.

Decarboxylation of amino acid is a key step for biosynthesis of several important cellular metabolites in the biological systems. This process is catalyzed by amino acid decarboxylases and most of them use pyridoxal-5'-phosphate (PLP) as a co-factor. PLP is bound to the active site of the enzyme by various interactions with the neighboring amino acid residues. In the present investigation, density functional theory (DFT) and real-time dynamics studies on both ligand-free and ligand-bound dopa decarboxylases (DDC) have been carried out in order to elucidate the factors responsible for facile decarboxylation and also for proper binding of PLP in the active site of the enzyme. It has been found that in the crystal structure Asp271 interacts with the pyridine nitrogen atom of PLP through H-bonding in both native and substrate-bound DDC. On the contrary, Thr246 is in close proximity to the oxygen of 3-OH of PLP pyridine ring only in the substrate-bound DDC. In the ligand-free enzyme, the distance between the oxygen atom of 3-OH group of PLP pyridine ring and oxygen atom of Thr246 hydroxyl group is not favorable for hydrogen bonding. Thus, present study reveals that hydrogen bonding with O3 of PLP with a hydrogen bond donor residue provided by the enzyme plays an important role in the decarboxylation process.

Suppression of VEGF and Decrease in Vascular Leakage by Pyridoxal 5'-Phosphate in Diabetic Rats

PURPOSE: The authors of the present study investigated whether pyridoxal 5'-phosphate (PLP), an active coenzyme of vitamin B6, could inhibit the development of diabetic retinopathy in streptozotocin (STZ)-induced diabetic rats. METHODS: Seven-week-old Spraque-Dawley rats (n = 20) were used in the present study. STZ (70 mg/kg) was injected intraperitoneally to induce diabetes. Blood glucose and body weight were monitored. Intraperitoneal injections of 5 microg and 50 microg PLP were administered every two days from the second week of induced diabetes. During the third week of PLP injections, the concentration level of plasma homocysteine was measured. In addition, functional status was examined by vitreous fluorophotometer and anatomical status by vascular endothelial growth factor (VEGF) staining in the retina. RESULTS: Based on vitreous fluorophotometry examination, the PLP injection group proved to have a lower level of fluorescein concentration in the vitreous. Additionally, immunohistochemical staining revealed down-regulation of VEGF expression in the PLP group. In addition, the PLP group had a lower plasma homocysteine concentration. However, an over-dosage injection of PLP did not appear to have any noticeable impact on the treatment of diabetes. CONCLUSIONS: PLP, an active coenzyme of vitamin B6, proved to have inhibitory effects on VEGF expression and vascular leakage in the diabetic rat retina.

Assessment of vitamin B6 status in Korean patients with newly diagnosed type 2 diabetes

The purpose of this study was to assess vitamin B6 intake and status in Korean patients with newly diagnosed type 2 diabetes. Sixty-four patients with newly diagnosed type 2 diabetes and 8-11% glycated hemoglobin (A1C), along with 28 age-matched non-diabetic subjects, participated. Dietary vitamin B6 intake was estimated by the 24 hour recall method and plasma pyridoxal 5'-phosphate (PLP) was measured. There was a significant difference in daily total calorie intake between the diabetic and non-diabetic groups (1,917 +/- 376 vs 2,093 +/- 311 kcal). There were no differences in intake of total vitamin B6 (2.51 +/- 0.91 vs 2.53 +/- 0.81 mg/d) or vitamin B6/1,000 kcal (1.31 +/- 0.42 vs 1.20 +/- 0.32 mg) between the diabetic and non-diabetic groups, andI intakes of total vitamin B6 were above the Korean RDA in both groups (180.0 +/- 57.9 vs 179.0 +/- 65.4). There was a higher percentage of diabetic subjects whose plasma PLP concentration was < 30 nmol/L compared to non-diabetic group. Plasma PLP levels tended to be lower in the diabetic subjects than in the non-diabetic subjects, although the difference was not statistically significant due to a large standard deviation (80.0 +/- 61.2 nmol/L vs 68.2 +/- 38.5 nmol/L). Nevertheless, plasma PLP levels should be monitored in pre-diabetic patients with diabetic risk factors as well as in newly diagnosed diabetic patients for long-term management of diabetes, even though this factor is not a major risk factor that contributes to the development of degenerative complications in certain patients.

Interaction of pyridoxal phosphate with the amino groups at the active site of 5-aminolevulinic acid dehydratase in maize.

Pyridoxal 5'-phosphate strongly and reversibly inhibited maize leaf 5-amino levulinic acid dehydratase. The inhibition was linearly competitive with respect to the substrate 5-aminolevulinic acid at pH values between 7 to 9·0. Pyridoxal was also effective as an inhibitor of the enzyme but pyridoxamine phosphate was not inhibitory. The results suggest that pyridoxal 5'-phosphate may be interacting with the enzyme either close to or at the 5- aminolevulinic acid binding site. This conclusion was further corroborated by the detection of a Schiff base between the enzyme and the substrate, 5-aminolevulinic acid and by reduction of pyridoxal phosphate and substrate complexes with sodium borohydride.

Metal binding to pyridoxal derivatives. An NMR study of the interaction of Eu(III) with pyridoxal phosphate and pyridoxamine phosphate.

The solution conformations of pyridoxal-5'-phosphate and pyridoxamine- 5'-phosphate have been investigated using Eu(III) as a nuclear magnetic resonance shift probe. Binding of Eu(III) to pyridoxal phosphate results in the formation of two complexes, at the phosphate group and the o-hydroxy-aldehyde moiety, which are in slow exchange on the nuclear magnetic resonance time-scale. The lanthanide-induced pseudo contact shifts calculated using the McConnell-Robertson equation (J. Chem. Soc. (1950), 22, 1561) are in good agreement with the experimentally observed values for both pyridoxal phosphate and pyridoxamine phosphate and lead to a family of closely related conformations.