Chemo-enzymatic synthesis of 3-(2-naphthyl)- L-alanine by an aminotransferase from the extreme thermophile, Thermococcus profundus

Hyper-thermostable aminotransferase from Thermococcus profundus (MsAT) was used to synthesize 3-(2-naphthyl)-l-alanine (Nal) by transamination between its corresponding -keto acid, 3-(2-naphthyl)pyruvate (NPA) and l-glutamate (Glu) at 70 °C. Equilibrium of this reaction was shifted toward Nal production due to its low solubility, giving rise to Nal precipitate. Optically pure Nal (>99% ee) was synthesized with 93% (mol mol^–1) yield from 180 mM NPA and 360 mM Glu.     

An approach for cloning biosynthetic genes of 2-deoxystreptamine-containing aminocyclitol antibiotics: isolation of a biosynthetic gene cluster of tobramycin from Streptomyces tenebrarius

Genes homologous to 2-deoxystreptamine (DOS) biosynthetic genes were isolated from aminoglycoside producers, Micromonospora and Streptomyces spp., using PCR primers based on the core sequences of 2-deoxy-scyllo-inosose (DOI) synthase and L-glutamine: scyllo-inosose aminotransferase (GIA). Identities of 40-45% were observed for DOI synthases, and 65-75% were observed for GIAs. The gene cluster of tobramycin biosynthesis was isolated from the genomic library of Streptomyces tenebrarius using DOI synthase as a probe. Sequencing of 33.9 kb revealed 24 putative open reading frames including the tobramycin biosynthetic gene cluster (13.8 kb) and a transport protein. This cluster encodes proteins homologous to 2-deoxystreptamine biosynthetic enzymes, glycosyltransferase and other aminocyclitols biosynthetic enzymes. Sequence analysis revealed the evolution of DOI synthases from 3-dehydroquinate (DHQ) synthases in actinomycetes. DOI synthases and GIA are therefore useful for cloning biosynthetic genes of DOS-containing aminocyclitol antibiotics or for screening such metabolites producers.     

Chlorophyll reduction in the seed of Brassica napus with a glutamate 1-semialdehyde aminotransferase antisense gene*

Chlorophyll reduction in the seed of Brassica can be achieved by downregulating its synthesis. To reduce chlorophyll synthesis, we have used a cDNA clone of Brassica napus encoding glutamate 1-semialdehyde aminotransferase (GSA-AT) to make an antisense construct for gene manipulation. Antisense glutamate 1-semialdehyde aminotransferase gene (Gsa) expression, directed by a Brassica napin promoter, was targeted specifically to the embryo of the developing seed. Transformants expressing antisense Gsa showed varying degrees of inhibition resulting in a range of chlorophyll reduction in the seeds. Seed growth and development were not affected by reduction of chlorophyll. Seeds from selfed transgenic plants germinated with high efficiency and growth of seedlings was vigorous. Seedlings from T₂ transgenic lines segregated into three distinctive phenotypes: dark green, light green and yellow, indicating the dominant inheritance of Gsa antisense gene. These transgenic lines have provided useful materials for the development of a low chlorophyll seed variety of B. napus.     

Arrest of chlorophyll accumulation prior to anthocyanin formation in Euphorbia pulcherrima

Euphorbia pulcherrima Klotz plants exposed to short days (11 h light/13 h dark) for a period of eight weeks, developed flowers and a red canopy consisting of bracts and few true leaves. Plants maintained for three weeks under short day conditions, failed to produce flower primordia or a red canopy in the following 5 weeks in continuous light. Between the 4th and 5th short day week, the youngest leaves began to accumulate anthocyanin and turned red while the apical meristems differentiated into flower primordia. Chlorophyll accumulation ceased at the onset of anthocyanin synthesis and the protein content per unit leaf area declined. mRNA for glutamyl-tRNA^Glu synthetase (EC 6.1.1.17) and glutamyl-tRNA^Glu reductase also declined during this period. Western blot analysis revealed a loss of glutamyl-tRNA^Glu reductase, glutamate 1-semialdehyde (EC 5.4.3.8) and the Mg-chelatase subunits, Olive and CH42, in the last 2 to 4 weeks of the photoperiod.     

Inhibition study of alanine aminotransferase enzyme using sequential online capillary electrophoresis analysis

We report the study of several inhibitors on alanine aminotransferase (ALT) enzyme using sequential online capillary electrophoresis (CE) assay. Using metal ions (Na⁺ and Mg²⁺) as example inhibitors, we show that evolution of the ALT inhibition reaction can be achieved by automatically and simultaneously monitoring the substrate consumption and product formation as a function of reaction time. The inhibition mechanism and kinetic constants of ALT inhibition with succinic acid and two traditional Chinese medicines were derived from the sequential online CE assay. Our study could provide valuable information about the inhibition reactions of ALT enzyme.     

Tonsil-derived mesenchymal stem cells alleviate concanavalin A-induced acute liver injury

Acute liver failure, the fatal deterioration of liver function, is the most common indication for emergency liver transplantation, and drug-induced liver injury and viral hepatitis are frequent in young adults. Stem cell therapy has come into the limelight as a potential therapeutic approach for various diseases, including liver failure and cirrhosis. In this study, we investigated therapeutic effects of tonsil-derived mesenchymal stem cells (T-MSCs) in concanavalin A (ConA)- and acetaminophen-induced acute liver injury. ConA-induced hepatitis resembles viral and immune-mediated hepatic injury, and acetaminophen overdose is the most frequent cause of acute liver failure in the United States and Europe. Intravenous administration of T-MSCs significantly reduced ConA-induced hepatic toxicity, but not acetaminophen-induced liver injury, affirming the immunoregulatory capacity of T-MSCs. T-MSCs were successfully recruited to damaged liver and suppressed inflammatory cytokine secretion. T-MSCs expressed high levels of galectin-1 and -3, and galectin-1 knockdown which partially diminished interleukin-2 and tumor necrosis factor α secretion from cultured T-cells. Galectin-1 knockdown in T-MSCs also reversed the protective effect of T-MSCs on ConA-induced hepatitis. These results suggest that galectin-1 plays an important role in immunoregulation of T-MSCs, which contributes to their protective effect in immune-mediated hepatitis. Further, suppression of T-cell activation by frozen and thawed T-MSCs implies great potential of T-MSC banking for clinical utilization in immune-mediated disease.     

Deletion of sterol O-acyltransferase 2 (SOAT2) function in mice deficient in lysosomal acid lipase (LAL) dramatically reduces esterified cholesterol sequestration in the small intestine and liver

Sterol O-acyltransferase 2 (SOAT2), also known as ACAT2, is the major cholesterol esterifying enzyme in the liver and small intestine (SI). Esterified cholesterol (EC) carried in certain classes of plasma lipoproteins is hydrolyzed by lysosomal acid lipase (LAL) when they are cleared from the circulation. Loss-of-function mutations in LIPA, the gene that encodes LAL, result in Wolman disease (WD) or cholesteryl ester storage disease (CESD). Hepatomegaly and a massive increase in tissue EC levels are hallmark features of both disorders. While these conditions can be corrected with enzyme replacement therapy, the question arose as to what effect the loss of SOAT2 function might have on tissue EC sequestration in LAL-deficient mice. When weaned at 21 days, Lal^−/−:Soat2^+/+ mice had a whole liver cholesterol content (mg/organ) of 24.7 mg vs 1.9 mg in Lal^+/+:Soat2^+/+ littermates, with almost all the excess sterol being esterified. Over the next 31 days, liver cholesterol content in the Lal^−/−:Soat2^+/+ mice increased to 145 ± 2 mg but to only 29 ± 2 mg in their Lal^−/−:Soat2^−/− littermates. The level of EC accumulation in the SI of the Lal^−/−:Soat2^−/− mice was also much less than in their Lal^−/−:Soat2^+/+ littermates. In addition, there was a >70% reduction in plasma transaminase activities in the Lal^−/−:Soat2^−/− mice. These studies illustrate how the severity of disease in a mouse model for CESD can be substantially ameliorated by elimination of SOAT2 function.     

Human liver peroxisomal alanine:glyoxylate aminotransferase: Different stability under chemical stress of the major allele,the minor allele,and its pathogenic G170R variant

The sensitivity to denaturant stress of the major (AGT-Ma) and the minor (AGT-Mi) allele of alanine:glyoxylate aminotransferase and P11L mutant has been examined by studying their urea-induced equilibrium unfolding processes with various spectroscopic and analytical techniques. AGT-Ma loses pyridoxal 5′-phosphate (PLP) and unfolds completely without exposing significant hydrophobic clusters through a two-state model (C_m ∼ 6.9 M urea). Instead, the unfolding of AGT-Mi and P11L variant proceeds in two steps. The first transition (C_m ∼ 4.6 M urea) involves PLP release, dimer dissociation and exposure of hydrophobic patches leading to a self-associated intermediate which is converted to an unfolded monomer in the second step. The unfolding pathways of apoAGT-Mi and apoP11L are similar to each other, but different from that of apoAGT-Ma. Notably, the monomerization step in apoAGT-Mi and apoP11L occurs with a C_m value (∼1.6 M urea) lower than in apoAGT-Ma (∼2.4 M urea). These data indicate that Pro11 is relevant for the stability of both the dimeric structure and the PLP binding site of AGT. Moreover, to understand the pathogenic consequences of G170R mutation on AGT-Mi at the protein level, G170R-Mi has been characterized. HoloG170R-Mi exhibits spectroscopic and catalytic features and urea unfolding profiles comparable to those of AGT-Mi, while the apo form monomerizes with a C_m of ∼1.1 M urea. These biochemical results are discussed in the light of the characteristics of the enzymatic phenotype of PH1 patients bearing G170R mutation in AGT-Mi and the positive response of these patients to pyridoxine treatment.     

Molecular architecture of DesV from Streptomyces venezuelae: a PLP-dependent transaminase involved in the biosynthesis of the unusual sugar desosamine

Desosamine is a 3-(dimethylamino)-3,4,6-trideoxyhexose found in certain macrolide antibiotics such as the commonly prescribed erythromycin. Six enzymes are required for its biosynthesis in Streptomyces venezuelae. The focus of this article is DesV, which catalyzes the PLP-dependent replacement of a 3-keto group with an amino functionality in the fifth step of the pathway. For this study the three-dimensional structures of both the internal aldimine and the ketimine intermediate with glutamate were determined to 2.05 A resolution. DesV is a homodimer with each subunit containing 12 alpha-helical regions and 12 beta-strands that together form three layers of sheet. The structure of the internal aldimine demonstrates that the PLP-cofactor is held in place by residues contributed from both subunits (Asp 164 and Gln 167 from Subunit I and Tyr 221 and Asn 235 from Subunit II). When the ketimine intermediate is present in the active site, the loop defined by Gln 225 to Ser 228 from Subunit II closes down upon the active site. The structure of DesV is similar to another sugar-modifying enzyme referred to as PseC. This enzyme is involved in the biosynthesis of pseudaminic acid, which is a sialic acid-like nonulosonate found in the flagellin of Helicobacter pylori. In the case of PseC, however, the amino group is transferred to the C-4 rather than the C-3 position. Details concerning the structural analysis of DesV and a comparison of its molecular architecture to that of PseC are presented.