• Molecules and Cells
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• v.46 no.12
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• pp.736-742
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• 2023

NifB, a radical S-adenosylmethionine (SAM) enzyme, is pivotal in the biosynthesis of the iron-molybdenum cofactor (FeMo-co), commonly referred to as the M-cluster. This cofactor, located within the active site of nitrogenase, is essential for the conversion of dinitrogen (N₂) to NH₃. Recognized as the most intricate metallocluster in nature, FeMo-co biosynthesis involves multiple proteins and a sequence of steps. Of particular significance, NifB directs the fusion of two [Fe₄S₄] clusters to assemble the 8Fe core, while also incorporating an interstitial carbide. Although NifB has been extensively studied, its molecular mechanisms remain elusive. In this review, we explore recent structural analyses of NifB and provide a comprehensive overview of the established catalytic mechanisms. We propose prospective directions for future research, emphasizing the relevance to biochemistry, agriculture, and environmental science. The goal of this review is to lay a solid foundation for future endeavors aimed at elucidating the atomic details of FeMo-co biosynthesis.

• Molecules and Cells
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• v.37 no.10
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• pp.719-726
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• 2014

The ${\gamma}$-Aminobutyric acid (GABA) that is found in prokaryotic and eukaryotic organisms has been used in various ways as a signaling molecule or a significant component generating metabolic energy under conditions of nutrient limitation or stress, through GABA catabolism. Succinic semialdehyde dehydrogenase (SSADH) catalyzes the oxidation of succinic semialdehyde to succinic acid in the final step of GABA catabolism. Here, we report the catalytic properties and two crystal structures of SSADH from Streptococcus pyogenes (SpSSADH) regarding its cofactor preference. Kinetic analysis showed that SpSSADH prefers $NADP^+$ over $NAD^+$ as a hydride acceptor. Moreover, the structures of SpSSADH were determined in an apo-form and in a binary complex with $NADP^+$ at $1.6{\AA}$ and $2.1{\AA}$ resolutions, respectively. Both structures of SpSSADH showed dimeric conformation, containing a single cysteine residue in the catalytic loop of each subunit. Further structural analysis and sequence comparison of SpSSADH with other SSADHs revealed that Ser158 and Tyr188 in SpSSADH participate in the stabilization of the 2'-phosphate group of adenine-side ribose in $NADP^+$. Our results provide structural insights into the cofactor preference of SpSSADH as the gram-positive bacterial SSADH.

• Journal of Microbiology and Biotechnology
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• v.16 no.9
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• pp.1434-1440
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• 2006

The stable anchorage of pyridoxal 5'-phosphate (PLP) in the active site of D-amino acid transaminase (D-AT) is crucial for the enzyme catalysis. The three-dimensional structure of D-AT revealed that Glu32 is one of the active site groups that may playa role in PLP binding. To prove the role of Glu32 in PLP stability, we firstly checked the rate of the potential rate-limiting step. The kinetic analysis showed that the rate of the ${\alpha}$-deprotonation step reduced to 26-folds in E32A mutant enzyme. Spectral analyses of the reaction of D-AT with D-serine revealed that the E32A mutant enzyme failed to stabilize the key enzyme-substrate intermediate, namely a quinonoid intermediate (E-Q). Finally, analysis of circular dichroism (CD) on the wild-type and E32A mutant enzymes showed that the optical activity of PLP in the enzyme active site was lost by the removal of the carboxylic group, proving that Glu32 is indeed involved in the cofactor anchorage. The results suggested that the electrostatic interaction network through the groups from PLP, Glu32, His47, and Arg50, which was observed from the three-dimensional structure of the enzyme, plays a crucial role in the stable anchorage of the cofactor to give necessary torsion to the plane of the cofactor-substrate complex.

• Journal of Korea Technical Association of The Pulp and Paper Industry
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• v.44 no.6
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• pp.43-49
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• 2012

In this work, the non-ionic polyethylene oxide(PEO)/phenolic formaldehyde resin(PFR) retention system, which was less affected by furnish charge, was analyzed for possibility and effectiveness when using recycled stock. When the micropolymer was added at the PEO/PFR retention system and the PAM/bentonite retention system, performance of retention and drainage was improved. When the cationic micropolymer was added on the PEO/PFR retention system, the highest retention was achieved. The Polyacrylamide(PAM)/bentonite system induced flocculation of white pitch by electrostatic flocculation. On the other hand, the non-ionic PEO/PFR retention system induced less flocculation of white pitch than the PAM/bentonite retention system.

• Journal of Microbiology and Biotechnology
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• v.20 no.6
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• pp.968-973
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• 2010

In order to study its biochemical properties, the integrase (IN) protein of feline foamy virus (FFV) was overexpressed in Escherichia coli, purified by two-step chromatography, (Talon column and heparin column), and characterized in biochemical aspects. For the three enzymatic reactions of the 3'-processing, strand transfer, and disintegration activities, the $Mn^{2+}$ ion was essentially required as a cofactor. Interestingly, $Co^{2+}$ and $Zn^{2+}$ ions were found to act as effective cofactors, whereas other transition elements such as $Ni^{2+}$, $Cu^{2+}$, $La^{3+}$, $Y^{3+}$, $Cd^{2+}$, $Li^{1+}$, $Ba^{2+}$, $Sr^{2+}$, and $V^{3+}$ were not. Regarding the substrate specificity, FFV IN has low substrate specificities as it cleaved in a significant level prototype foamy virus (PFV) U5 LTR substrate as well as FFV U5 LTR substrate, whereas PFV IN did not. Finally, the 3'-processing activity was observed in high concentrations of several solvents such as CHAPS, glycerol, Tween 20, and Triton X-100, which are generally used for dissolution of chemicals in inhibitor screening. Therefore, in this first report showing its biochemical properties, FFV IN is proposed to have low specificities on the use of cofactor and substrate for enzymatic reaction as compared with other retroviral INs.

• BMB Reports
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• v.44 no.5
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• pp.341-346
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• 2011

The single-stranded DNA binding activity of the Escherichia coli RecA protein is crucial for homologous recombination to occur. This and other biochemical activities of ssDNA binding proteins may be affected by various factors. In this study, we analyzed the effect of $CaCl_2$, NaCl and $NH_4NO_3$ salts in combination with the pH and nucleotide cofactor effect on the ssDNA-binding activity of RecA. The studies revealed that, in addition to the inhibitory effect, these salts exert also a stimulatory effect on RecA. These effects occur only under very strict conditions, and the presence or absence and the type of nucleotide cofactor play here a major role. It was observed that in contrast to ATP, ATP${\gamma}$S prevented the inhibitory effect of NaCl and $NH_4NO_3$, even at very high salt concentration. These results indicate that ATP${\gamma}$S most likely stabilizes the structure of RecA required for DNA binding, making it resistant to high salt concentrations.

• Journal of Microbiology and Biotechnology
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• v.23 no.2
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• pp.218-224
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• 2013

The aldo-keto reductases catalyze reduction reactions using various aliphatic and aromatic aldehydes/ketones. Most reductases require NADPH exclusively as their cofactors. However, NADPH is much more expensive and unstable than NADH. In this study, we attempted to change the five amino acid residues that interact with the 2'-phosphate group of the adenosine ribose of NADPH. These residues were selected based on a docking model of the YOL151W reductase and were substituted with other amino acids to develop NADH-utilizing enzymes. Ten mutants were constructed by site-directed mutagenesis and expressed in Escherichia coli. Among them, four mutants showed higher reductase activities than wild-type when using the NADH cofactor. Analysis of the kinetic parameters for the wild type and mutants indicated that the $k_{cat}/K_{m}$ value of the Asn9Glu mutant toward NADH increased 3-fold. A docking model was used to show that the carboxyl group of Glu 9 of the mutant formed an additional hydrogen bond with the 2'-hydroxyl group of adenosine ribose. The Asn9Glu mutant was able to produce (R)-ethyl-4-chloro-3-hydroxyl butanoate rapidly when using the NADH regeneration system.

• Current Research on Agriculture and Life Sciences
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• v.32 no.4
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• pp.199-202
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• 2014

The bioconversion of cellulosic biomass hydrolyzates consisting mainly of glucose and xylose requires the use of engineered Saccharomyces cerevisiae expressing a heterologous xylose pathway. However, there is concern that a fungal xylose pathway consisting of NADPH-specific xylose reductase (XR) and $NAD^+$-specific xylitol dehydrogenase (XDH) may result in a cellular redox imbalance. However, the glycerol biosynthesis and glycerol degradation pathways of S. cerevisiae, termed here as the glycerol cycle, has the potential to balance the cofactor requirements for xylose metabolism, as it produces NADPH by consuming NADH at the expense of one mole of ATP. Therefore, this study tested if the glycerol cycle could improve the xylose metabolism of engineered S. cerevisiae by cofactor balancing, as predicted by an in-silico analysis using elementary flux mode (EFM). When the GPD1 gene, the first step of the glycerol cycle, was overexpressed in the XR/XDH-expressing S. cerevisiae, the glycerol production significantly increased, while the xylitol and ethanol yields became negligible. The reduced xylitol yield suggests that enough $NAD^+$ was supplied for XDH by the glycerol cycle. However, the GPD1 overexpression completely shifted the carbon flux from ethanol to glycerol. Thus, moderate expression of GPD1 may be necessary to achieve improved ethanol production through the cofactor balancing.

• Journal of Microbiology and Biotechnology
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• v.29 no.4
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• pp.607-616
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• 2019

In this study, functionalized poplar powder (FPP) was used as a support material for the immobilization of enoate reductase (ER) and glucose-6-phosphate dehydrogenase (GDH) by covalent binding. Under optimal conditions, the immobilization efficiency of ER-FPP and GDH-FPP was 95.1% and 84.7%, and the activity recovery of ER and GDH was 47.5% and 37.8%, respectively. Scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) analysis indicated that FPP was a suitable carrier for enzyme immobilization. ER-FPP and GDH-FPP exhibit excellent thermal stabilities and superior reusability. Especially, ER-FPP and GDH-FPP enable the continuous conversion of 4-(4-Methoxyphenyl)-3-buten-2-one with $NAD^+$ recycling. While the immobilization strategies established here were simple and inexpensive, they exploited a new method for the immobilization and application of ER and its cofactor recycling system.

• Applied Biological Chemistry
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• v.37 no.3
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• pp.203-209
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• 1994

When $^{14}C-{\alpha}-endosulfan$ was incubated with carp liver, kidney and gut preparations, it was metabolized to water soluble and organosoluble metabolites. In an in vitro test, endosulfan was converted to endosulfan ${\alpha}-hydroxyether$ (EHE), endosulfan alcohol (EA) and endosulfan ether (EE). The addition of NADPH resulted in rapid conversion of endosulfan to the metabolites in 105,000 g soluble fraction and microsomes. However, the rate of metabolism of endosulfan in liver, kidney and gut supplemented with NADPH as a cofactor was higher in the 105,000 g soluble fraction than that in the microsomes of carp under incubation conditions. The enzymes probably involved in the metabolism of endosulfan include the glutathione S-transferase (GST) and the mixed function oxidases (MFO), based on the evidence that addition of either GSH or NADPH increased the degradation of endosulfan.