The molecular basis of the nonprocessive elongation mechanism in levansucrases

Levansucrases (LSs) synthesize levan, a β2-6-linked fructose polymer, by successively transferring the fructosyl moiety from sucrose to a growing acceptor molecule. Elucidation of the levan polymerization mechanism is important for using LSs in the production of size-defined products for application in the food and pharmaceutical industries. For a deeper understanding of the levan synthesis reaction, we determined the crystallographic structure of Bacillus subtilis LS (SacB) in complex with a levan-type fructooligosaccharide (FOS) and utilized site-directed mutagenesis to identify residues involved in substrate binding. The presence of a levanhexaose molecule in the central catalytic cavity allowed us to identify five substrate-binding subsites (-1, +1, +2, +3, and +4). Mutants affecting residues belonging to the identified acceptor subsites showed similar substrate affinity (Km) values to the wild type (WT) Km value but had a lower turnover number and transfructosylation/hydrolysis ratio. Most importantly, compared to the WT, the variants progressively yielded smaller-sized low-molecular weight (LMW) levans, as the affected subsites that were closer to the catalytic site, but without affecting their ability to synthesized high-molecular weight (HMW) levans. Furthermore, an additional oligosaccharide-binding (OB) site 20 Å away from the catalytic pocket was identified,and its potential participation in the elongation mechanism is discussed. Our results clarify, for the first time, the interaction of the enzyme with an acceptor/product oligosaccharide and elucidate the molecular basis of the nonprocessive levan elongation mechanism of LSs.

Download Full-text

Functional definition of the tobacco protoporphyrinogen IX oxidase substrate-binding site

Biochemical Journal ◽

10.1042/bj20061321 ◽

2007 ◽

Vol 402 (3) ◽

pp. 575-580 ◽

Cited By ~ 29

Author(s):

Ilka U. Heinemann ◽

Nina Diekmann ◽

Ava Masoumi ◽

Michael Koch ◽

Albrecht Messerschmidt ◽

...

Keyword(s):

Chlorophyll Biosynthesis ◽

Substrate Binding ◽

Protoporphyrin Ix ◽

Site Directed Mutagenesis ◽

Amino Acid Residues ◽

Enzyme Defects ◽

Km Value ◽

Function Relationship ◽

Definition Of

PPO (protoporphyrinogen IX oxidase) catalyses the flavin-dependent six-electron oxidation of protogen (protoporphyrinogen IX) to form proto (protoporphyrin IX), a crucial step in haem and chlorophyll biosynthesis. The apparent Km value for wild-type tobacco PPO2 (mitochondrial PPO) was 1.17 μM, with a Vmax of 4.27 μM·min−1·mg−1 and a catalytic activity kcat of 6.0 s−1. Amino acid residues that appear important for substrate binding in a crystal structure-based model of the substrate docked in the active site were interrogated by site-directed mutagenesis. PPO2 variant F392H did not reveal detectable enzyme activity indicating an important role of Phe392 in substrate ring A stacking. Mutations of Leu356, Leu372 and Arg98 increased kcat values up to 100-fold, indicating that the native residues are not essential for establishing an orientation of the substrate conductive to catalysis. Increased Km values of these PPO2 variants from 2- to 100-fold suggest that these residues are involved in, but not essential to, substrate binding via rings B and C. Moreover, one prominent structural constellation of human PPO causing the disease variegate porphyria (N67W/S374D) was successfully transferred into the tobacco PPO2 background. Therefore tobacco PPO2 represents a useful model system for the understanding of the structure–function relationship underlying detrimental human enzyme defects.

Download Full-text

Aromatic residues within the substrate-binding cleft of Bacillus circulans chitinase A1 are essential for hydrolysis of crystalline chitin

Biochemical Journal ◽

10.1042/bj20030419 ◽

2003 ◽

Vol 376 (1) ◽

pp. 237-244 ◽

Cited By ~ 84

Author(s):

Takeshi WATANABE ◽

Yumiko ARIGA ◽

Urara SATO ◽

Tadayuki TORATANI ◽

Masayuki HASHIMOTO ◽

...

Keyword(s):

Catalytic Domain ◽

Substrate Binding ◽

Substrate Inhibition ◽

Site Directed Mutagenesis ◽

Bacillus Circulans ◽

Aromatic Residues ◽

Km Value ◽

Chitin Hydrolysis ◽

Binding Cleft ◽

Hydrolysis Of

Bacillus circulans chitinase A1 (ChiA1) has a deep substrate-binding cleft on top of its (β/α)8-barrel catalytic domain and an interaction between the aromatic residues in this cleft and bound oligosaccharide has been suggested. To study the roles of these aromatic residues, especially in crystalline-chitin hydrolysis, site-directed mutagenesis of these residues was carried out. Y56A and W53A mutations at subsites −5 and −3, respectively, selectively decreased the hydrolysing activity against highly crystalline β-chitin. W164A and W285A mutations at subsites +1 and +2, respectively, decreased the hydrolysing activity against crystalline β-chitin and colloidal chitin, but enhanced the activities against soluble substrates. These mutations increased the Km-value when reduced (GlcNAc)5 (where GlcNAc is N-acetylglucosamine) was used as the substrate, but decreased substrate inhibition observed with wild-type ChiA1 at higher concentrations of this substrate. In contrast with the selective effect of the other mutations, mutations of W433 and Y279 at subsite −1 decreased the hydrolysing activity drastically against all substrates and reduced the kcat-value, measured with 4-methylumbelliferyl chitotrioside to 0.022% and 0.59% respectively. From these observations, it was concluded that residues Y56 and W53 are only essential for crystalline-chitin hydrolysis. W164 and W285 are very important for crystalline-chitin hydrolysis and also participate in hydrolysis of other substrates. W433 and Y279 are both essential for catalytic reaction as predicted from the structure.

Download Full-text

Structural studies of myo-inositol kinase

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s205327331409528x ◽

2014 ◽

Vol 70 (a1) ◽

pp. C471-C471

Author(s):

Ryuhei Nagata ◽

Masahiro Fujihashi ◽

Takaaki Sato ◽

Haruyuki Atomi ◽

Kunio Miki

Keyword(s):

Substrate Binding ◽

Complex Structure ◽

Site Directed Mutagenesis ◽

Hydroxyl Group ◽

Structural Studies ◽

Binding Mechanism ◽

Structure Comparison ◽

Substrate Complex ◽

Chromatography Analysis ◽

Km Value

The TK2285 protein from a hyperthermopilic archaeon Thermococcus kodakarensis is a myo-inositol kinase. Only two myo-inositol kinases have been identified so far. One is the TK2285 protein and the other is an enzyme from Zea mays. Both of them synthesize myo-inositol monophosphate that shows enantiomerism. Because it is too difficult to discriminate enantiomers by NMR or chromatography analysis, it has not been identified which of the six hydroxyls is phosphorylated by these enzymes. Also, little is known about the substrate recognition of myo-inositol kinase, since only the unliganded crystal structure of TK2285 has been reported. In order to reveal the substrate-binding mechanism of myo-inositol kinase and identify the phosphorylated hydroxyl group of the product, we determined the crystal structures of TK2285 as the substrate-complex and the product-complex. The substrate-complex of TK2285 was prepared by using the TK2285, myo-inositol and AMP-PCP, and the products-complex was prepared by incubating the TK2285 with myo-inositol and ATP. The substrate-complex structure showed that all of the six hydroxyls of myo-inositol interacted with TK2285. This coincides with the fact that the Km value for myo-inositol is 100-1000 fold lower than those for other sugars. Also 3-hydroxyl group of myo-inositol, which the gamma-phosphate of AMP-PCP was nearest to, was thought to be phosphorylated by this enzyme. This was proved by the product-complex structure that had ADP and myo-inositol 3-phosphate. Site-directed mutagenesis and structure comparison with TK2285 homologs also provided information about the substrate-binding mechanism of myo-inositol kinase.

Download Full-text

Structural contributions of Delta class glutathione transferase active-site residues to catalysis

Biochemical Journal ◽

10.1042/bj20091939 ◽

2010 ◽

Vol 428 (1) ◽

pp. 25-32 ◽

Cited By ~ 14

Author(s):

Jantana Wongsantichon ◽

Robert C. Robinson ◽

Albert J. Ketterman

Keyword(s):

Crystal Structures ◽

Glutathione Transferase ◽

Substrate Binding ◽

Conformational Stability ◽

Site Directed Mutagenesis ◽

Active Site Residues ◽

Molecular Rearrangement ◽

Hydrophobic Substrate ◽

Substrate Processing ◽

First Time

GST (glutathione transferase) is a dimeric enzyme recognized for biotransformation of xenobiotics and endogenous toxic compounds. In the present study, residues forming the hydrophobic substrate-binding site (H-site) of a Delta class enzyme were investigated in detail for the first time by site-directed mutagenesis and crystallographic studies. Enzyme kinetics reveal that Tyr111 indirectly stabilizes GSH binding, Tyr119 modulates hydrophobic substrate binding and Phe123 indirectly modulates catalysis. Mutations at Tyr111 and Phe123 also showed evidence for positive co-operativity for GSH and 1-chloro-2,4-dinitrobenzene respectively, strongly suggesting a role for these residues in manipulating subunit–subunit communication. In the present paper we report crystal structures of the wild-type enzyme, and two mutants, in complex with S-hexylglutathione. This study has identified an aromatic ‘zipper’ in the H-site contributing a network of aromatic π–π interactions. Several residues of the cluster directly interact with the hydrophobic substrate, whereas others indirectly maintain conformational stability of the dimeric structure through the C-terminal domain (domain II). The Y119E mutant structure shows major main-chain rearrangement of domain II. This reorganization is moderated through the ‘zipper’ that contributes to the H-site remodelling, thus illustrating a role in co-substrate binding modulation. The F123A structure shows molecular rearrangement of the H-site in one subunit, but not the other, explaining weakened hydrophobic substrate binding and kinetic co-operativity effects of Phe123 mutations. The three crystal structures provide comprehensive evidence of the aromatic ‘zipper’ residues having an impact upon protein stability, catalysis and specificity. Consequently, ‘zipper’ residues appear to modulate and co-ordinate substrate processing through permissive flexing.

Download Full-text

Comprehensive in silico Study of GLUT10: Prediction of Possible Substrate Binding Sites and Interacting Molecules

Current Pharmaceutical Biotechnology ◽

10.2174/1389201020666190613152030 ◽

2020 ◽

Vol 21 (2) ◽

pp. 117-130 ◽

Cited By ~ 1

Author(s):

Mohammad J. Hosen ◽

Mahmudul Hasan ◽

Sourav Chakraborty ◽

Ruhshan A. Abir ◽

Abdullah Zubaer ◽

...

Keyword(s):

Virtual Screening ◽

Binding Site ◽

Glucose Transporter ◽

Substrate Binding ◽

Mutation Screening ◽

Homology Model ◽

Connective Tissue Disorder ◽

Crystallographic Structure ◽

Substrate Binding Site

Objectives: The Arterial Tortuosity Syndrome (ATS) is an autosomal recessive connective tissue disorder, mainly characterized by tortuosity and stenosis of the arteries with a propensity towards aneurysm formation and dissection. It is caused by mutations in the SLC2A10 gene that encodes the facilitative glucose transporter GLUT10. The molecules transported by and interacting with GLUT10 have still not been unambiguously identified. Hence, the study attempts to identify both the substrate binding site of GLUT10 and the molecules interacting with this site. Methods: As High-resolution X-ray crystallographic structure of GLUT10 was not available, 3D homology model of GLUT10 in open conformation was constructed. Further, molecular docking and bioinformatics investigation were employed. Results and Discussion: Blind docking of nine reported potential in vitro substrates with this 3D homology model revealed that substrate binding site is possibly made with PRO531, GLU507, GLU437, TRP432, ALA506, LEU519, LEU505, LEU433, GLN525, GLN510, LYS372, LYS373, SER520, SER124, SER533, SER504, SER436 amino acid residues. Virtual screening of all metabolites from the Human Serum Metabolome Database and muscle metabolites from Human Metabolite Database (HMDB) against the GLUT10 revealed possible substrates and interacting molecules for GLUT10, which were found to be involved directly or partially in ATS progression or different arterial disorders. Reported mutation screening revealed that a highly emergent point mutation (c. 1309G>A, p. Glu437Lys) is located in the predicted substrate binding site region. Conclusion: Virtual screening expands the possibility to explore more compounds that can interact with GLUT10 and may aid in understanding the mechanisms leading to ATS.

Download Full-text

Antimutagenic, Antiproliferative and Antioxidant Properties of Sea Grape Leaf Extract Fractions (Coccoloba uvifera L.)

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520621999210104201242 ◽

2021 ◽

Vol 21 ◽

Author(s):

Jorge A. Ramos-Hernández ◽

Montserrat Calderón-Santoyo ◽

Armando Burgos-Hernández ◽

Joel S. García- Romo ◽

Arturo Navarro-Ocaña ◽

...

Keyword(s):

Antioxidant Properties ◽

In Vitro Tests ◽

Antimutagenic Activity ◽

Hexane Extraction ◽

Biological Potential ◽

Hct 116 ◽

Pharmaceutical Industries ◽

Grape Leaf ◽

First Time

Background: Cancer is a disease characterized by the invasion and uncontrolled growth of cells. One of the best ways to minimize the harmful effects of mutagens is through the use of natural antimutagens. In this regard, the search for new antimutagens that act in the chemoprevention could represent a promising field in this area. Objective: In this study biological potential of 11 fractions from Coccoloba uvifera L. leaf hexane extract was evaluated by several in vitro tests. Methods: Leaves were lyophilized and hexane extraction was performed. The extract was fractionated by column chromatography with hexane, ethyl acetate, and methanol. The antimutagenic (Ames test), antiproliferative (MTT test), and antioxidant capacity (DPPH, ABTS, and ferrous ion chelation) of the fractions were evaluated. Results: Fractions 4, 6, 8, and 9 have antimutagenic activity (against sodium azide in strain TA100), fraction 11 showed antiproliferative capacity (IC50 of 24 ± 9 μg/mL in cells of HCT 116). The fractions with the highest activity were analyzed by HPLC-MS and lupeol, acacetin, and β-sitosterol were identified. Conclusion: This study demonstrates, for the first time, the bioactivity of C. uvifera leaf as a new source of high biological value compounds (HBVC), which can be of interest to the food and pharmaceutical industries.

Download Full-text

Chitosanase from Streptomyces sp. strain N174: a comparative review of its structure and function

Biochemistry and Cell Biology ◽

10.1139/o97-079 ◽

1997 ◽

Vol 75 (6) ◽

pp. 687-696 ◽

Cited By ~ 20

Author(s):

Tamo Fukamizo ◽

Ryszard Brzezinski

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Substrate Binding ◽

Structure And Function ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Amino Acid Residues ◽

Streptomyces Sp ◽

Binding Cleft ◽

And Function

Novel information on the structure and function of chitosanase, which hydrolyzes the beta -1,4-glycosidic linkage of chitosan, has accumulated in recent years. The cloning of the chitosanase gene from Streptomyces sp. strain N174 and the establishment of an efficient expression system using Streptomyces lividans TK24 have contributed to these advances. Amino acid sequence comparisons of the chitosanases that have been sequenced to date revealed a significant homology in the N-terminal module. From energy minimization based on the X-ray crystal structure of Streptomyces sp. strain N174 chitosanase, the substrate binding cleft of this enzyme was estimated to be composed of six monosaccharide binding subsites. The hydrolytic reaction takes place at the center of the binding cleft with an inverting mechanism. Site-directed mutagenesis of the carboxylic amino acid residues that are conserved revealed that Glu-22 and Asp-40 are the catalytic residues. The tryptophan residues in the chitosanase do not participate directly in the substrate binding but stabilize the protein structure by interacting with hydrophobic and carboxylic side chains of the other amino acid residues. Structural and functional similarities were found between chitosanase, barley chitinase, bacteriophage T4 lysozyme, and goose egg white lysozyme, even though these proteins share no sequence similarities. This information can be helpful for the design of new chitinolytic enzymes that can be applied to carbohydrate engineering, biological control of phytopathogens, and other fields including chitinous polysaccharide degradation. Key words: chitosanase, amino acid sequence, overexpression system, reaction mechanism, site-directed mutagenesis.

Download Full-text

Specific Seminal Plasma Fractions Are Responsible for the Modulation of Sperm–PMN Binding in the Donkey

Animals ◽

10.3390/ani11051388 ◽

2021 ◽

Vol 11 (5) ◽

pp. 1388

Author(s):

Jordi Miró ◽

Jaime Catalán ◽

Henar Marín ◽

Iván Yánez-Ortiz ◽

Marc Yeste

Keyword(s):

Molecular Weight ◽

Sperm Motility ◽

Seminal Plasma ◽

Potential Effect ◽

Polymorphonuclear Neutrophils ◽

Low Fertility ◽

Plasma Fractions ◽

Complex Fluid ◽

First Time

While artificial insemination (AI) with frozen-thawed sperm results in low fertility rates in donkeys, the addition of seminal plasma, removed during cryopreservation, partially counteracts that reduction. Related to this, an apparent inflammatory reaction in jennies is induced following AI with frozen-thawed sperm, as a high amount of polymorphonuclear neutrophils (PMN) are observed within the donkey uterus six hours after AI. While PMN appear to select the sperm that ultimately reach the oviduct, two mechanisms, phagocytosis and NETosis, have been purported to be involved in that clearance. Remarkably, sperm interacts with PMN, but the presence of seminal plasma reduces that binding. As seminal plasma is a complex fluid made up of different molecules, including proteins, this study aimed to evaluate how different seminal plasma fractions, separated by molecular weight (<3, 3–10, 10–30, 30–50, 50–100, and >100 kDa), affect sperm–PMN binding. Sperm motility, viability, and sperm–PMN binding were evaluated after 0 h, 1 h, 2 h, 3 h, and 4 h of co-incubation at 38 °C. Two seminal plasma fractions, including 30–50 kDa or 50–100 kDa proteins, showed the highest sperm motility and viability. As viability of sperm not bound to PMN after 3 h of incubation was the highest in the presence of 30–50 and 50–100 kDa proteins, we suggest that both fractions are involved in the control of the jenny’s post-breeding inflammatory response. In conclusion, this study has shown for the first time that specific fractions rather than the entire seminal plasma modulate sperm–PMN binding within the donkey uterus. As several proteins suggested to be involved in the control of post-AI endometritis have a molecular weight between 30 and 100 kDa, further studies aimed at determining the identity of these molecules and evaluating their potential effect in vivo are much warranted.

Download Full-text

Microbial N2O consumption in and above marine N2O production hotspots

The ISME Journal ◽

10.1038/s41396-020-00861-2 ◽

2020 ◽

Author(s):

Xin Sun ◽

Amal Jayakumar ◽

John C. Tracey ◽

Elizabeth Wallace ◽

Colette L. Kelly ◽

...

Keyword(s):

Kinetic Parameters ◽

Substrate Affinity ◽

N2o Production ◽

Anoxic Waters ◽

Production And Consumption ◽

Consumption Rates ◽

Anoxic Zones ◽

First Time ◽

Microbial N

AbstractThe ocean is a net source of N2O, a potent greenhouse gas and ozone-depleting agent. However, the removal of N2O via microbial N2O consumption is poorly constrained and rate measurements have been restricted to anoxic waters. Here we expand N2O consumption measurements from anoxic zones to the sharp oxygen gradient above them, and experimentally determine kinetic parameters in both oxic and anoxic seawater for the first time. We find that the substrate affinity, O2 tolerance, and community composition of N2O-consuming microbes in oxic waters differ from those in the underlying anoxic layers. Kinetic parameters determined here are used to model in situ N2O production and consumption rates. Estimated in situ rates differ from measured rates, confirming the necessity to consider kinetics when predicting N2O cycling. Microbes from the oxic layer consume N2O under anoxic conditions at a much faster rate than microbes from anoxic zones. These experimental results are in keeping with model results which indicate that N2O consumption likely takes place above the oxygen deficient zone (ODZ). Thus, the dynamic layer with steep O2 and N2O gradients right above the ODZ is a previously ignored potential gatekeeper of N2O and should be accounted for in the marine N2O budget.

Download Full-text

Enhancing the thermostability and activity of uronate dehydrogenase from Agrobacterium tumefaciens LBA4404 by semi-rational engineering

Bioresources and Bioprocessing ◽

10.1186/s40643-019-0267-3 ◽

2019 ◽

Vol 6 (1) ◽

Cited By ~ 1

Author(s):

Hui-Hui Su ◽

Fei Peng ◽

Pei Xu ◽

Xiao-Ling Wu ◽

Min-Hua Zong ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Rational Design ◽

Glucuronic Acid ◽

Specific Activity ◽

Value Added ◽

Catalytic Efficiency ◽

Site Directed Mutagenesis ◽

Glucaric Acid ◽

Triple Mutant ◽

Pharmaceutical Industries

Abstract Background Glucaric acid, one of the aldaric acids, has been declared a “top value-added chemical from biomass”, and is especially important in the food and pharmaceutical industries. Biocatalytic production of glucaric acid from glucuronic acid is more environmentally friendly, efficient and economical than chemical synthesis. Uronate dehydrogenases (UDHs) are the key enzymes for the preparation of glucaric acid in this way, but the poor thermostability and low activity of UDH limit its industrial application. Therefore, improving the thermostability and activity of UDH, for example by semi-rational design, is a major research goal. Results In the present work, three UDHs were obtained from different Agrobacterium tumefaciens strains. The three UDHs have an approximate molecular weight of 32 kDa and all contain typically conserved UDH motifs. All three UDHs showed optimal activity within a pH range of 6.0–8.5 and at a temperature of 30 °C, but the UDH from A. tumefaciens (At) LBA4404 had a better catalytic efficiency than the other two UDHs (800 vs 600 and 530 s−1 mM−1). To further boost the catalytic performance of the UDH from AtLBA4404, site-directed mutagenesis based on semi-rational design was carried out. An A39P/H99Y/H234K triple mutant showed a 400-fold improvement in half-life at 59 °C, a 5 °C improvement in $$ {\text{T}}_{ 5 0}^{ 1 0} $$ T 50 10 value and a 2.5-fold improvement in specific activity at 30 °C compared to wild-type UDH. Conclusions In this study, we successfully obtained a triple mutant (A39P/H99Y/H234K) with simultaneously enhanced activity and thermostability, which provides a novel alternative for the industrial production of glucaric acid from glucuronic acid.

Download Full-text