Aims: Heterologous holoenzyme formation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) has been a challenge due to a limited understanding of its biogenesis. Unlike bacterial Rubiscos, eukaryotic Rubiscos are incompatible with the Escherichia coli (E. coli) chaperone system to fold and assemble into the functional hexadecameric conformation (L8S8), which comprises eight large subunits (RbcL) and eight small subunits (RbcS). Our previous study reported three sections (residues 248-297, 348-397 and 398-447) within the RbcL of Synechococcus elongatus PCC6301, which may be important for the formation of L8S8 in E. coli. The present study further examined these three sections separately, dividing them into six sections of 25 residues (i.e., residues 248-272, 273-297, 348-372, 373-397, 398-422 and 423-447). Methodology and results: Six chimeric Rubiscos with each section within the RbcL from Synechococcus replaced by their respective counterpart sequence from Chlamydomonas reinhardtii were constructed and checked for their effect on holoenzyme formation in E. coli. The present study shows that Section 1 (residues 248-272; section of Synechococcus RbcL replaced by corresponding Chlamydomonas sequence), Section 2 (residues 273-297), Section 3 (residues 348-372) and Section 6 (residues 423-447) chimeras failed to fold and assemble despite successful expression of both RbcL and RbcS. Only Section 4 (residues 373-397) and 5 (residues 398-422) chimeras could form L8S8 in E. coli. Conclusion, significance and impact of study GroEL chaperonin mediates the folding of bacterial RbcL in E. coli. Therefore, residues 248-297, 348-372 and 423-447 of Synechococcus RbcL may be important for interacting with the GroEL chaperonin for successful holoenzyme formation in E. coli.
Synechococcus ; Ribulose-Bisphosphate Carboxylase ; Escherichia coli ; Holoenzymes

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.
Escherichia coli/metabolism* ; Carboxy-Lyases/metabolism* ; Catalysis ; Cadaverine/metabolism*

Objective: Propionic acidemia is a rare inherited metabolic disorder caused by propionyl CoA carboxylase (PCC) deficiency. This study aims to analyze the clinical characteristics and gene variations of Chinese patients with propionic acidemia, and to explore the correlation between clinical phenotypes and genotypes. Methods: Single-center, retrospective and observational study. Seventy-eight patients of propionic acidemia (46 males and 32 females) from 20 provinces and autonomous regions were admitted from January 2007 to April 2022. Their age of initial diagnosis ranged from 7 days to 15 years. The clinical manifestations, biochemical and metabolic abnormalities, genetic variations, diagnosis, treatment and outcome were studied. Chi-Square test or Mann-Whitney U test were used for statistical analysis. Results: Among 78 cases, 6 (7.7%) were identified by newborn screening; 72 (92.3%) were clinically diagnosed after onset, and the age of onset was 2 hours after birth to 15 years old; 32 cases had early-onset disease and 40 cases had late-onset disease. The initial manifestations included lethargy, hypotonia, vomiting, feeding difficulties, developmental delay, epilepsy, and coma. Among the 74 cases who accepted gene analysis, 35 (47.3%) had PCCA variants and 39 (52.7%) had PCCB variants. A total of 39 PCCA variants and 32 PCCB variants were detected, among which c.2002G>A and c.229C>T in PCCA and c.838dupC and c.1087T>C in PCCB were the most common variants in this cohort. The variants c.1228C>T and c.1283C>T in PCCB may be related to early-onset type. The variants c.838dupC, c.1127G>T and c.1316A>G in PCCB, and c.2002G>A in PCCA may be related to late-onset disease. Six patients detected by newborn screening and treated at asymptomatic stage developed normal. The clinically diagnosed 72 cases had varied complications. 10 (12.8%) cases of them died. 62 patients improved after metabolic therapy by L-carnitine and diet. Six patients received liver transplantation because of recurrent metabolic crisis. Their clinical symptoms were markedly improved. Conclusion: The clinical manifestations of propionic acidemia are complex and lack of specificity. Newborn screening and high-risk screening are keys for early treatment and better outcome. The correlation between the genotype and phenotype of propionic acidemia is unclear, but certain variants may be associated with early-onset or late-onset propionic acidemia.
Carnitine ; Female ; Genotype ; Humans ; Male ; Methylmalonyl-CoA Decarboxylase/metabolism* ; Mutation ; Phenotype ; Propionic Acidemia/genetics* ; Retrospective Studies

Ornithine decarboxylase (ODC) is a key enzyme in the biosynthetic pathway of polyamines and catalyzes the decarboxylation of ornithine to produce putrescine. Inhibition of ODC activity is a potential approach for the prevention and treatment of many diseases including cancer, as the expression levels and the activities of ODC in many abnormal cells and tumor cells are generally higher than those of normal cells. The discovery and evaluation of ODC inhibitors rely on the monitoring of the reaction processes catalyzed by ODC. There are several commonly used methods for analyzing the activity of ODC, such as measuring the yield of putrescine by high performance liquid chromatography, or quantifying the yield of isotope labelled carbon dioxide. However, the cumbersome operation and cost of these assays, as well as the difficulty to achieve high-throughput and real-time detection, hampered their applications. In this work, we optimized a real-time label-free method for analyzing the activity of ODC based on the macromolecule cucurbit[6]uril (CB6) and a fluorescent dye, DSMI (trans-4-[4-(dimethylamino) styryl]-1-methylpyridinium iodide). Finally, the optimized method was used to determine the activities of different ODC inhibitors with different inhibition mechanisms.
Bridged-Ring Compounds ; Imidazoles ; Ornithine ; Ornithine Decarboxylase ; Ornithine Decarboxylase Inhibitors ; Putrescine

Terpenoid indole (TIAs) and β-carboline alkaloids (BCAs), such as suppressant reserpine, vasodilatory yohimbine, and antimalarial quinine, are natural compounds derived from strictosidine. These compounds can exert powerful pharmacological effects but be obtained from limited source in nature. the whole biosynthetic pathway of TIAs and BCAs, The Pictet-Spengler reaction catalyzed by strictosidine synthase (STR; EC: 4.3.3.2) is the rate-limiting step. Therefore, it is necessary to investigate their biosynthesis pathways, especially the role of STR, and related findings will support the biosynthetic generation of natural and unnatural compounds. This review summarizes the latest studies concerning the function of STR in TIA and BCA biosynthesis, and illustrates the compounds derived from strictosidine. The substrate specificity of STR based on its structure is also summarized. Proteins that contain six-bladed four-stranded β-propeller folds in many organisms, other than plants, are listed. The presence of these folds may lead to similar functions among organisms. The expression of STR gene can greatly influence the production of many compounds. STR is mainly applied to product various valuable drugs in plant cell suspension culture and biosynthesis in other carriers.
Alkaloids/biosynthesis* ; Carbolines/metabolism* ; Carbon-Nitrogen Lyases ; Indoles/metabolism* ; Terpenes/metabolism*

A boy attended the hospital at the age of 1 month due to left hand tremor for 1 week. A blood test showed a reduction in serum uric acid and a cranial MRI showed encephalomalacia, atrophy, and cystic changes. The boy had microcephalus, unusual facial features (long face, long forehead, protruded forehead, long philtrum, low nasal bridge, facial swelling, and thick lower lip), hypertonia of lower extremities, and severe global developmental delay. Whole-exome sequencing performed for the boy detected a homozygous mutation, c.217C > T(p.R73W), in the
Carbon-Carbon Lyases ; China ; Humans ; Infant, Newborn ; Male ; Metal Metabolism, Inborn Errors ; Mutation ; Uric Acid

Dopamine is the precursor of a variety of natural antioxidant compounds. In the body, dopamine acts as a neurotransmitter that regulates a variety of physiological functions of the central nervous system. Thus, dopamine is used for the clinical treatment of various types of shock. Dopamine could be produced by engineered microbes, but with low efficiency. In this study, DOPA decarboxylase gene from Sus scrofa (Ssddc) was cloned into plasmids with different copy numbers, and transformed into a previously developed L-DOPA producing strain Escherichia coli T004. The resulted strain was capable of producing dopamine from glucose directly. To further improve the production of dopamine, a sequence-based homology alignment mining (SHAM) strategy was applied to screen more efficient DOPA decarboxylases, and five DOPA decarboxylase genes were selected from 100 candidates. In shake-flask fermentation, the DOPA decarboxylase gene from Homo sapiens (Hsddc) showed the highest dopamine production (3.33 g/L), while the DOPA decarboxylase gene from Drosophila Melanogaster (Dmddc) showed the least residual L-DOPA concentration (0.02 g/L). In 5 L fed-batch fermentations, production of dopamine by the two engineered strains reached 13.3 g/L and 16.2 g/L, respectively. The residual concentrations of L-DOPA were 0.45 g/L and 0.23 g/L, respectively. Finally, the Ssddc and Dmddc genes were integrated into the genome of E. coli T004 to obtain genetically stable dopamine-producing strains. In 5 L fed-batch fermentation, 17.7 g/L of dopamine was produced, which records the highest titer reported to date.
Animals ; Dopa Decarboxylase/genetics* ; Dopamine/biosynthesis* ; Drosophila melanogaster/genetics* ; Escherichia coli/metabolism* ; Humans ; Metabolic Engineering

carboxylase. Most cases are diagnosed after the detection of metabolic abnormalities—such as hyperammonemia, metabolic acidosis, and ketosis—associated with complaints of vomiting, feeding difficulties, and hypotonia during the neonatal period. However, in rare late-onset cases, mild or vague symptoms make the diagnosis more challenging. Even though acute pancreatitis is relatively uncommon in children, it can occur in association with PA. We present the case of a 4-year-old child who was admitted owing to the complaint of recurrent pancreatitis and had not previously been diagnosed with having metabolic disease. During inpatient treatment for acute pancreatitis, convulsions occurred with concomitant hyperammonemia, metabolic acidosis, coagulopathy, and shock 1 week after the administration of total parenteral nutrition. He was diagnosed to have PA after a metabolic work-up and confirmed to have novel mutation by molecular genetic analysis. Because children with PA may have acute pancreatitis, although rare, vomiting and abdominal pain should raise a suspicion of acute pancreatitis. On the contrary, even among children who have never been diagnosed with a metabolic disease, if a child has recurrent pancreatitis, metabolic pancreatitis caused by organic acidemia should be considered.]]>
Abdominal Pain ; Acidosis ; Child ; Child, Preschool ; Diagnosis ; Diethylpropion ; Humans ; Hyperammonemia ; Inpatients ; Metabolic Diseases ; Methylmalonyl-CoA Decarboxylase ; Molecular Biology ; Muscle Hypotonia ; Pancreatitis ; Parenteral Nutrition, Total ; Propionic Acidemia ; Seizures ; Shock ; Vomiting

The hallmark of cisplatin-induced acute kidney injury is the necrotic cell death in the kidney proximal tubules. However, an effective approach to limit cisplatin nephrotoxicity remains unknown. Spermidine is a polyamine that protects against oxidative stress and necrosis in aged yeasts, and the present study found that exogenous spermidine markedly attenuated tubular necrosis and kidney dysfunction, but not apoptosis, during cisplatin nephrotoxicity. In addition, exogenous spermidine potently inhibited oxidative/nitrative DNA damage, poly(ADP-ribose) polymerase 1 (PARP1) activation and ATP depletion after cisplatin injection. Conversely, inhibition of ornithine decarboxylase (ODC) via siRNA transfection in vivo significantly increased DNA damage, PARP1 activation and ATP depletion, resulting in acceleration of tubular necrosis and kidney dysfunction. Finally, exogenous spermidine removed severe cisplatin injury induced by ODC inhibition. In conclusion, these data suggest that spermidine protects kidneys against cisplatin injury through DNA damage and tubular necrosis, and this finding provides a novel target to prevent acute kidney injury including nephrotoxicity.
Acceleration ; Acute Kidney Injury ; Adenosine Triphosphate ; Apoptosis ; Cell Death ; Cisplatin* ; DNA Damage ; Kidney ; Lipid Peroxidation ; Necrosis* ; Ornithine Decarboxylase ; Oxidative Stress ; Poly(ADP-ribose) Polymerases ; RNA, Small Interfering ; Spermidine* ; Transfection ; Yeasts

In plants, lipoxygenases (LOXs) play a crucial role in biotic and abiotic stresses. In our previous study, five 13-LOX genes of oriental melon were regulated by abiotic stress but it is unclear whether the 9-LOX is involved in biotic and abiotic stresses. The promoter analysis revealed that CmLOX09 (type of 9-LOX) has hormone elements, signal substances, and stress elements. We analyzed the expression of CmLOX09 and its downstream genes-CmHPL and CmAOS-in the leaves of four-leaf stage seedlings of the oriental melon cultivar "Yumeiren" under wound, hormone, and signal substances. CmLOX09, CmHPL, and CmAOS were all induced by wounding. CmLOX09 was induced by auxin (indole acetic acid, IAA) and gibberellins (GA₃); however, CmHPL and CmAOS showed differential responses to IAA and GA₃. CmLOX09, CmHPL, and CmAOS were all induced by hydrogen peroxide (H₂O₂) and methyl jasmonate (MeJA), while being inhibited by abscisic acid (ABA) and salicylic acid (SA). CmLOX09, CmHPL, and CmAOS were all induced by the powdery mildew pathogen Podosphaera xanthii. The content of 2-hexynol and 2-hexenal in leaves after MeJA treatment was significantly higher than that in the control. After infection with P. xanthii, the diseased leaves of the oriental melon were divided into four levels-levels 1, 2, 3, and 4. The content of jasmonic acid (JA) in the leaves of levels 1 and 3 was significantly higher than that in the level 0 leaves. In summary, the results suggested that CmLOX09 might play a positive role in the response to MeJA through the hydroperoxide lyase (HPL) pathway to produce C6 alcohols and aldehydes, and in the response to P. xanthii through the allene oxide synthase (AOS) pathway to form JA.
Abscisic Acid ; Acetates/chemistry* ; Aldehyde-Lyases/metabolism* ; Aldehydes/chemistry* ; Cucurbitaceae/genetics* ; Cyclopentanes/chemistry* ; Cytochrome P-450 Enzyme System/metabolism* ; Gene Expression Profiling ; Gene Expression Regulation, Plant ; Hormones/metabolism* ; Hydrogen Peroxide/metabolism* ; Intramolecular Oxidoreductases/metabolism* ; Lipoxygenase/metabolism* ; Oxylipins/chemistry* ; Plant Leaves/genetics* ; Plant Proteins/metabolism* ; Promoter Regions, Genetic ; Salicylic Acid/chemistry* ; Seedlings/metabolism* ; Signal Transduction ; Stress, Physiological ; Transgenes