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Based on previous studies in bacteria and eukaryotes, lipoate-protein ligases (Lpls) have been considered to be involved exclusively in lipoate salvage. The genetic analyses in this study on the lipoate-protein ligase in T. kodakarensis , however, suggest otherwise and that the enzyme is additionally involved in de novo protein lipoylation.

Lipoic acid is a sulfur-containing cofactor and a component of the glycine cleavage system (GCS) involved in C(1) compound metabolism and the 2-oxoacid dehydrogenases that catalyze the oxidative decarboxylation of 2-oxoacids. Lipoic acid is found in all domains of life and is generally synthesized as a lipoyl group on the H-protein of the GCS or the E2 subunit of 2-oxoacid dehydrogenases. Lipoyl synthase catalyzes the insertion of two sulfur atoms to the C-6 and C-8 carbon atoms of the octanoyl moiety on the octanoyl-H-protein or octanoyl-E2 subunit. Although the hyperthermophilic archaeon Thermococcus kodakarensis seemed able to synthesize lipoic acid, a classical lipoyl synthase (LipA) gene homolog cannot be found on the genome. In this study, we aimed to identify the lipoyl synthase in this organism. Genome information analysis suggested that the TK2109 and TK2248 genes, which had been annotated as biotin synthase (BioB), are both involved in lipoic acid metabolism. Based on the chemical reaction catalyzed by BioB, we predicted that the genes encode proteins that catalyze the lipoyl synthase reaction. Genetic analysis of TK2109 and TK2248 provided evidence that these genes are involved in lipoic acid biosynthesis. The purified TK2109 and TK2248 recombinant proteins exhibited lipoyl synthase activity toward a chemically synthesized octanoyl-octapeptide. These in vivo and in vitro analyses indicated that the TK2109 and TK2248 genes encode a structurally novel lipoyl synthase. TK2109 and TK2248 homologs are widely distributed among the archaeal genomes, suggesting that in addition to the LipA homologs, the two proteins represent a new group of lipoyl synthases in archaea. IMPORTANCE Lipoic acid is an essential cofactor for GCS and 2-oxoacid dehydrogenases, and α-lipoic acid has been utilized as a medicine and attracted attention as a supplement due to its antioxidant activity. The biosynthesis pathways of lipoic acid have been established in Bacteria and Eucarya but not in Archaea. Although some archaeal species, including Sulfolobus, possess a classical lipoyl synthase (LipA) gene homolog, many archaeal species, including T. kodakarensis, do not. In addition, the biosynthesis mechanism of the octanoyl moiety, a precursor for lipoyl group biosynthesis, is also unknown for many archaea. As the enzyme identified in T. kodakarensis most likely represents a new group of lipoyl synthases in Archaea, the results obtained in this study provide an important step in understanding how lipoic acid is synthesized in this domain and how the two structurally distinct lipoyl synthases evolved in nature.

2-Chloronicotinic acid (2-CA) is an important building block for a series of agrochemicals and pharmaceuticals. Amidase-catalyzed hydrolysis of 2-chloronicotinamide is one of the most attractive approaches for 2-CA production. However, development of the bioprocess was plagued by low activity of amidase for 2-chloronicotinamide. In this work, an amidase signature (AS) family amidase from Pantoea sp. (Pa-Ami), with superior activity for nicotinamide and its chlorinated derivatives, was exploited and characterized. Kinetic analysis and molecular docking clearly indicated that chlorine substitution in the pyridine ring of nicotinamide, especially the substitution at 2-position led to a dramatic decrease of Pa-Ami activity. The productivity of the bioprocess was significantly improved using fed-batch mode at low reaction temperature and 2-CA was produced as high as 370 mM with a substrate conversion of 94.2%. These results imply that Pa-Ami is potentially promising biocatalyst for industrial production of 2-CA.

2-Chloronicotinic acid is a key intermediate of pharmaceuticals and pesticides. Amidase-catalyzed hydrolysis provides a promising enzymatic method for 2-chloronicotinic acid production from 2-chloronicotinamide. However, biocatalytic hydrolysis of 2-chloronicotinamide is difficult due to the strong steric and electronic effect caused by 2-position chlorine substituent of the pyridine ring. In this study, an amidase from a Pantoea sp. (Pa-Ami) was designed and engineered to have improved catalytic properties. Single mutant G175A and double mutant G175A/A305T strains exhibited 3.2- and 3.7-fold improvements in their specific activity for 2-chloronicotinamide, and the catalytic efficiency was significantly increased, with kcat/Km values 3.1 and 10.0 times higher than that of the wild type, respectively. Structure-function analysis revealed that the distance between Oγ of Ser177 (involved in the catalytic triad) and the carbonyl carbon of 2-chloronicotinamide was shortened in the G175A mutant, making the nucleophilic attack on the Oγ of Ser177 easier by virtue of proper orientation. In addition, the A305T mutation contributed to a suitable tunnel formation to facilitate the substrate entry and product release, resulting in improved catalytic efficiency. With the G175A/A305T double mutant as a biocatalyst, a maximum of 1,220 mM 2-chloronicotinic acid was produced with a 94% conversion, and the space-time yield reached as high as 575 gproduct liter−1 day−1. These results provide not only a novel robust biocatalyst for the production of 2-chloronicotinic acid but also new insights into amidase structure-function relationships. IMPORTANCE In recent years, the demand for 2-chloronicotinic acid has been greatly increased. To date, several chemical methods have been used for the synthesis of 2-chloronicotinic acid, but all include tedious steps and/or drastic reaction conditions, resulting in both economic and environmental issues. It is requisite to develop an efficient and green synthesis route. We recently screened Pa-Ami and demonstrated its potential for synthesis of 2-chloronicotinic acid from 2-chloronicotinamide. However, chlorine substitution on the pyridine ring of nicotinamide significantly affected the activity of Pa-Ami. Especially for 2-chloronicotinamide, the enzyme activity and catalytic efficiency were relatively low. In this study, based on structure-function analysis, we succeeded in engineering the amidase by structure-guided saturation mutagenesis. The engineered Pa-Ami exhibited quite high catalytic activity toward 2-chloronicotinamide and could serve as a promising biocatalyst for the biosynthesis of 2-chloronicotinic acid.

1-Cyanocyclohexaneacetic acid is the key intermediate of gabapentin. A novel bioprocess catalyzed by amidase was developed for efficient production of 1-cyanocyclohexaneacetic acid from 1-cyanocyclohexaneacetamide, which can be prepared with high efficiency by nitrile hydratase-catalyzed regioselective hydration of 1-cyanocyclohexaneacetonitrile. Kinetic analysis and molecular docking of three recombinant amidase demonstrated that amidase (Pa-Ami) from Pantoea sp. was the most robust biocatalyst for hydrolysis of 1-cyanocyclohexaneacetamide with the kcat/Km value of 208.2 ± 16.2 mM−1 s−1. Some key parameters of the bioprocess, such as substrate loading, catalyst loading and product inhibition, were investigated. Enzymatic hydrolysis of 80 g/L of 1-cyanocyclohexaneacetamide was completed within 20 min using 1 g/L wet whole cells of recombinant Escherichia coli BL21, leading to high ratio of substrate to catalyst (S/C-ratio, 80) and high space-time yield (5794.7 gproduct L−1 d−1). These encouraging results indicated the great potential of Pa-Ami in practical production of gabapentin.

Demyelination contributes to the functional deficits after spinal cord injury (SCI). Therefore, remyelination may be an important strategy to facilitate repair after SCI. Oligodendrocyte precursor cells (OPCs) are immature oligodendrocytes and can differentiate into myelin-forming cells of central nervous system under certain conditions. OPC transplantation is an attractive approach for the treatment of demyelinating diseases. In this study, we transplanted OPCs expressing green fluorescent protein (GFP-OPCs) into normal and injured rat spinal cords to evaluate the differentiation of transplanted OPCs in vivo. Unfortunately, the grafted GFP-OPCs, in spinal cord whether normal or injured, were all differentiated into astrocytes, but not oligodendrocytes. Our further study indicated that inflammatory environment might not be the key factor influencing the differentiation of OPCs. Some spinal cord components, such as bone morphogenetic proteins (BMPs), were the major factors that induced OPCs to differentiate into astrocytes. The three types of BMP receptor (BMPRIA, IB and II) could all be detected in OPCs, and the astroglial differentiation of OPCs induced by spinal cord homogenate extract (SCHE) in vitro could be blocked partly by noggin, an antagonist of BMP. These results suggested that the BMPR signal transduction pathway might be one of the key factors which determine the differentiation direction of engrafted OPCs in spinal cord.

The expression of major histocompatibility complex (MHC) antigens on neural stem cells (NSCs) and their lineages is tightly related to the fate of these cells as grafts in allogenic transplantation. In this study, we observed that NSCs derived from embryonic rat forebrain expressed MHC class I and class II molecules at a low level, whereas the cells differentiated from NSCs, including neurons, astrocytes, and oligodendrocytes, lost their MHC expression. However, a proinflammatory factor, interferon-gamma (IFN-gamma), could induce and up-regulate the expression of MHC in both NSCs and their differentiated lineages in vitro. These results suggest that predifferentiating NSCs into lineage-limited cells prior to transplantation combined with controlling the local production of proinflammatory cytokines moderately may potentially benefit the survival of transplants.

The xeroderma pigmentosum complementation group G (XPG) gene plays an important role in the DNA nucleotide excision repair (NER) pathway. Several studies have investigated the association between the XPG Asp1104His polymorphism and breast cancer; however, the results have been inconsistent. Therefore, we conducted a meta-analysis of 8 published articles (10 case-control studies) including a total of 5,235 patients with breast cancer and 5,685 healthy controls. The results demonstrated that the XPG Asp1104His polymorphism was not associated with breast cancer in the overall population [His vs. Asp, odds ratio (OR)=1.00, 95% confidence interval (CI): 0.91-1.08; His/His vs. Asp/Asp, OR=0.96, 95% CI: 0.83-1.11; Asp/His vs. Asp/Asp, OR=1.02, 95% CI: 0.94-1.11; His/His+Asp/His vs. Asp/Asp, OR=1.03, 95% CI: 0.92-1.15; and His/His vs. Asp/Asp+Asp/His, OR=0.93, 95% CI: 0.81-1.06]. In the subgroup analysis by ethnicity, no significant association was observed in European subjects. In conclusion, this meta-analysis suggested that the XPG Asp1104His polymorphism is not associated with breast cancer risk.

Conditioned medium obtained from B104 neuroblastoma cells (B104CM) has been used widely for inducing oligodendrocyte progenitor cells (OPCs) from neural precursor cells (NPCs). Our previous studies have demonstrated that E16 rat spinal cord-derived NPCs could be induced to differentiate into OPCs using a combination of B104CM and basic fibroblast growth factor (bFGF). Here we report the development of a more efficient and reliable approach to generate large quantities of highly purified OPCs from spinal cord-derived NPCs using a combination of platelet derived growth factor (PDGF) and bFGF. We demonstrated that, after the two factors application, over 90% cells displayed typical bipolar or tripolar morphology and expressed markers for OPCs including A2B5 (90.36 +/- 4.59%), NG2 (93.63 +/- 3.37%) and platelet derived growth factor alpha receptor (PDGFR; 90.35 +/- 1.95%). Our results indicated that the PDGF/bFGF combination is more efficient in generating OPCs than the B104CM/bFGF. And it is a more potent combination of factors in promoting proliferation of OPCs.

We have previously established a culture method to isolate and cultivate neural stem cells (NSCs) derived from the rat embryonic brain and spinal cord. In the present study, we demonstrate that the spinal cord-derived NSCs can be induced to differentiate into oligodendrocyte precursor cells (OPCs) with a combined treatment composed of (1) conditioned medium collected from B104 neuroblastoma cells (B104CM) and (2) basic fibroblast growth factor (bFGF, 10 ng/ml). After induction, over 95% of the cells displayed bipolar or tri-polar morphology and expressed A2B5 and platelet derived growth factor receptor-alpha (PDGFR-alpha), markers that are specific for OPCs. Among PDGFR-alpha positive OPCs, only a few cells expressed glia fibrillary acidic protein (GFAP) and none expressed beta-tubulin III. In the presence of B104CM and bFGF, OPCs proliferated rapidly, formed spheres, expanded for multiple passages, and maintained their phenotypic properties. Upon withdrawal of B104CM and bFGF, these cells differentiated into either O4/GlaC-positive oligodendrocytes (OLs) or GFAP- and A2B5-positive type-2 astrocytes. Our results indicate that NSCs can be induced to differentiate into OPCs that possess properties of self-renewal and differentiation into oligodendrocytes and type-2 astrocytes, a property similar to that of O-2A progenitor cells. The OPCs can be maintained in an undifferentiated state over multiple divisions as long as both B104CM and bFGF are present in the medium. Thus, large quantity of OPCs can be obtained through this method for potential therapeutical interventions for various neurological degenerative diseases.

AIM: To study the relation between CYP1A1 Ile462Val polymorphism and colorectal cancer risk by meta-analysis. METHODS: A meta-analysis was performed to investigate the relation between CYP1A1 Ile462Val polymorphism and colorectal cancer risk by reviewing the related studies until September 2010. Data were extracted and analyzed. Crude odds ratio (OR) with 95% confidence interval (CI) was used to assess the strength of relation between CYP1A1 Ile462Val polymorphism and colorectal cancer risk. RESULTS: Thirteen published case-control studies including 5336 cases and 6226 controls were acquired. The pooled OR with 95% CI indicated that CYP1A1 Ile462Val polymorphism was significantly related with colorectal cancer risk (Val/Val vs Ile/Ile: OR = 1.47, 95% CI: 1.16-1.86, P = 0.002; dominant model: OR = 1.33, 95% CI: 1.01-1.75, P = 0.04; recessive model: OR = 1.49, 95% CI: 1.18-1.88, P = 0.0009). Subgroup ethnicity analysis showed that CYP1A1 Ile462Val polymorphism was also significantly related with colorectal cancer risk in Europeans (Ile/Val vs Ile/Ile: OR = 1.22, 95% CI: 1.05-1.42, P = 0.008; dominant model: OR = 1.24, 95% CI: 1.07-1.43, P = 0.004) and Asians (Val/Val vs Ile/Ile: OR = 1.40, 95% CI: 1.07-1.82, P = 0.01; recessive model: OR = 1.46, 95% CI: 1.12-1.89, P = 0.005). CONCLUSION: CYP1A1 Ile462Val may be an increased risk factor for colorectal cancer.