scholarly journals Biochemical characterization of a family IV esterase with R-form enantioselectivity from a compost metagenomic library

2021 ◽  
Vol 64 (1) ◽  
Author(s):  
Jong Eun Park ◽  
Geum Seok Jeong ◽  
Hyun Woo Lee ◽  
Hoon Kim
Keyword(s):  
Biochemical Characterization ◽  
Metagenomic Library ◽  
Hormone Sensitive Lipase ◽  
Size Exclusion ◽  
Potential Candidate ◽  
Multiple Sequence ◽  
Exclusion Chromatography ◽  
Dimeric Form ◽  
Triton X ◽  
Family Iv

AbstractA novel family IV esterase (hormone-sensitive lipase, HSL) gene, est15L, was isolated from a compost metagenomic library. Encoded Est15L comprised 328 amino acids with a molecular weight of 34,770 kDa and was an intracellular esterase without a signal peptide. The multiple sequence alignment (MSA) of Est15L with other family IV esterases showed conserved regions such as HGG, DYR, GXSXG, DPL, and GXIH. Native Est15L was a dimeric form from the results of size exclusion chromatography. It was optimally active at 50 ℃ and pH 9.0, indicating alkaline esterase. However, it showed a low thermostability with half-lives of 30.3 at 30 ℃ and 2.7 min at 40 ℃. It preferred p-nitrophenyl butyrate (C4) with Km and Vmax values of 0.28 mM and 270.8 U/mg, respectively. Est15L was inhibited by organic solvents such as 30% methanol, isopropanol, and acetonitrile with residual activities of 12.5, 0.9, and 0.3%, respectively. It was also inhibited by 1% SDS and 1% PMSF; however, Est15L maintained its activity at 1% Triton X-100 and EDTA. Est15L was inhibited by Cu2+, Zn2+, Mn2+, Co2+, Fe2+, and Na+. In addition, Est15L hydrolyzed glyceryl tributyrate with a residual substrate amount of 43.7% at 60 min but could not hydrolyze the oils (fish and olive) and glyceryl trioleate. Interestingly, Est15L showed significant enantioselectivity toward the R-form with a residual substrate amount of 44.6%, lower than that of the S-form (83.5%). Considering its properties, Est15L can be a potential candidate for chemical reactions, such as the synthesis of pharmaceutical compounds.

scholarly journals Structural and Biochemical Characterization of a Cold-Active PMGL3 Esterase with Unusual Oligomeric Structure

Biomolecules ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 57
Author(s):  
Konstantin M. Boyko ◽  
Mariya V. Kryukova ◽  
Lada E. Petrovskaya ◽  
Elena A. Kryukova ◽  
Alena Y. Nikolaeva ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
3D Structure ◽  
Hormone Sensitive Lipase ◽  
Size Exclusion ◽  
Wild Type ◽  
Prolonged Incubation ◽  
Gene Coding ◽  
Cold Active ◽  
Tetrameric Structure ◽  
Exclusion Chromatography

The gene coding for a novel cold-active esterase PMGL3 was previously obtained from a Siberian permafrost metagenomic DNA library and expressed in Escherichia coli. We elucidated the 3D structure of the enzyme which belongs to the hormone-sensitive lipase (HSL) family. Similar to other bacterial HSLs, PMGL3 shares a canonical α/β hydrolase fold and is presumably a dimer in solution but, in addition to the dimer, it forms a tetrameric structure in a crystal and upon prolonged incubation at 4 °C. Detailed analysis demonstrated that the crystal tetramer of PMGL3 has a unique architecture compared to other known tetramers of the bacterial HSLs. To study the role of the specific residues comprising the tetramerization interface of PMGL3, several mutant variants were constructed. Size exclusion chromatography (SEC) analysis of D7N, E47Q, and K67A mutants demonstrated that they still contained a portion of tetrameric form after heat treatment, although its amount was significantly lower in D7N and K67A compared to the wild type. Moreover, the D7N and K67A mutants demonstrated a 40 and 60% increase in the half-life at 40 °C in comparison with the wild type protein. Km values of these mutants were similar to that of the wt PMGL3. However, the catalytic constants of the E47Q and K67A mutants were reduced by ~40%.

Molecular cloning and biochemical characterization of a NAD-dependent sorbitol dehydrogenase from cold-adapted Pseudomonas mandelii

2021 ◽  
Author(s):  
Quynh DangThu ◽  
Thu-Thuy Nguyen ◽  
Sei-Heon Jang ◽  
ChangWoo Lee
Keyword(s):  
Biochemical Characterization ◽  
Size Exclusion ◽  
Sorbitol Dehydrogenase ◽  
High Catalytic Activity ◽  
Active Site Residues ◽  
Tertiary Structures ◽  
Cold Adapted ◽  
Pseudomonas Mandelii ◽  
Catalytic Active Site ◽  
Exclusion Chromatography

Abstract Sugar alcohols (polyols) have important roles as nutrients, anti-freezing agents, and scavengers of free radicals in cold-adapted bacteria, but the characteristics of polyol dehydrogenases in cold-adapted bacteria remain largely unknown. In this study, based on the observation that a cold-adapted bacterium Pseudomonas mandelii JR-1 predominantly utilized D-sorbitol as its carbon source, among the four polyols examined (D-galactitol, D-mannitol, D-sorbitol, or D-xylitol), we cloned and characterized a sorbitol dehydrogenase (SDH, EC 1.1.1.14) belonging to the short-chain dehydrogenase/reductase family from this bacterium (the SDH hereafter referred to as PmSDH). PmSDH contained Asn111, Ser140, Tyr153, and Lys157 as catalytic active site residues and existed as a ∼67 kDa dimer in size-exclusion chromatography. PmSDH converted D-sorbitol to D-fructose using NAD+ as a coenzyme and, vice versa, D-fructose to D-sorbitol using NADH as a coenzyme. PmSDH maintained its conformational flexibility, secondary and tertiary structures, and thermal stability at 4–25°C. At 40°C, PmSDH was rapidly denatured. These results indicate that PmSDH, which has a flexible structure and a high catalytic activity at colder temperatures, is well-suited to sorbitol utilization in the cold-adapted bacterium P. mandelii JR-1.

scholarly journals Molecular cloning and biochemical characterization of rabbit factor XI

2002 ◽  
Vol 367 (1) ◽  
pp. 49-56 ◽  
Author(s):  
Dipali SINHA ◽  
Mariola MARCINKIEWICZ ◽  
David GAILANI ◽  
Peter N. WALSH
Keyword(s):  
Human Factor ◽  
Biochemical Characterization ◽  
Protein A ◽  
Size Exclusion ◽  
Factor Xi ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Intracellular Process ◽  
And Function

Human factor XI, a plasma glycoprotein required for normal haemostasis, is a homodimer (160kDa) formed by a single interchain disulphide bond linking the Cys-321 of each Apple 4 domain. Bovine, porcine and murine factor XI are also disulphide-linked homodimers. Rabbit factor XI, however, is an 80kDa polypeptide on non-reducing SDS/PAGE, suggesting that rabbit factor XI exists and functions physiologically either as a monomer, as does prekallikrein, a structural homologue to factor XI, or as a non-covalent homodimer. We have investigated the structure and function of rabbit factor XI to gain insight into the relation between homodimeric structure and factor XI function. Characterization of the cDNA sequence of rabbit factor XI and its amino acid translation revealed that in the rabbit protein a His residue replaces the Cys-321 that forms the interchain disulphide linkage in human factor XI, explaining why rabbit factor XI is a monomer in non-reducing SDS/PAGE. On size-exclusion chromatography, however, purified plasma rabbit factor XI, like the human protein and unlike prekallikrein, eluted as a dimer, demonstrating that rabbit factor XI circulates as a non-covalent dimer. In functional assays rabbit factor XI and human factor XI behaved similarly. Both monomeric and dimeric factor XI were detected in extracts of cells expressing rabbit factor XI. We conclude that the failure of rabbit factor XI to form a covalent homodimer due to the replacement of Cys-321 with His does not impair its functional activity because it exists in plasma as a non-covalent homodimer and homodimerization is an intracellular process.

scholarly journals Purification and partial biochemical characterization of normal human interleukin 1.

1984 ◽  
Vol 160 (3) ◽  
pp. 772-787 ◽  
Author(s):  
J A Schmidt
Keyword(s):  
High Performance ◽  
Biochemical Characterization ◽  
Interleukin 1 ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Thymocyte Proliferation ◽  
Exclusion Chromatography ◽  
Normal Human

A protocol for the rapid, efficient purification of the major charged species of human interleukin 1 (IL-1) has been developed using high performance anion exchange and size exclusion chromatography. The isolated material is pure as determined by sodium dodecyl sulfate (SDS) gradient polyacrylamide gel electrophoresis (PAGE) and analytical isoelectric focusing (IEF). The molecular weight of the purified material is 15,000 and the isoelectric point (pI) is 6.8, values that are in good agreement with those previously reported for human IL-1. 10(-10) M concentrations of the purified material give half-maximal stimulation in the thymocyte proliferation assay. Amounts of IL-1 sufficient for receptor studies and detailed biochemical analysis can now be produced on a regular basis.

scholarly journals Physicochemical Evidence that Treponema pallidum TroA Is a Zinc-Containing Metalloprotein That Lacks Porin-Like Structure

1999 ◽  
Vol 181 (14) ◽  
pp. 4420-4423 ◽  
Author(s):  
Ranjit K. Deka ◽  
Yong-Hwan Lee ◽  
Kayla E. Hagman ◽  
Dmitriy Shevchenko ◽  
Clifford A. Lingwood ◽  
...  
Keyword(s):  
Signal Sequence ◽  
Treponema Pallidum ◽  
Structural Features ◽  
Size Exclusion ◽  
Leader Peptide ◽  
Stoichiometric Ratio ◽  
Phase Partitioning ◽  
X Ray Scattering ◽  
Exclusion Chromatography ◽  
Triton X

ABSTRACT Although TroA (Tromp1) was initially reported to be aTreponema pallidum outer membrane protein with porin-like properties, subsequent studies have suggested that it actually is a periplasmic substrate-binding protein involved in the transport of metals across the treponemal cytoplasmic membrane. Here we conducted additional physicochemical studies to address the divergent viewpoints concerning this protein. Triton X-114 phase partitioning of recombinant TroA constructs with or without a signal sequence corroborated our prior contention that the native protein’s amphiphilic behavior is due to its uncleaved leader peptide. Whereas typical porins are trimers with extensive β-barrel structure, size exclusion chromatography and circular dichroism spectroscopy revealed that TroA was a monomer and predominantly alpha-helical. Neutron activation, atomic absorption spectroscopy, and anomalous X-ray scattering all demonstrated that TroA binds zinc in a 1:1 molar stoichiometric ratio. TroA does not appear to possess structural features consistent with those of bacterial porins.

scholarly journals Biochemical Characterization of a Group-5 Soluble Diiron Monooxygenase Hydroxylase and Related Chaperonin-like Component

2020 ◽  
Author(s):  
Chihiro Inoue ◽  
Yoshitaka Abe ◽  
Nobutaka Fujieda
Keyword(s):  
Biochemical Characterization ◽  
Quaternary Structure ◽  
Expression System ◽  
Functional Expression ◽  
Size Exclusion ◽  
Native Page ◽  
Dinuclear Iron ◽  
Exclusion Chromatography ◽  
Group 5

<p>Recently, the functional expression of group-5 hydroxylase component (MimA and MimC) in <i>Escherichia coli </i>along with its related chaperonin-like component (MimG) was reported by Furuya and Kino. In this study, we report the purification via a heterologous expression system and the biochemical characterization of MimAC, the complex of MimA and MimC and MimG to understand their exact roles. MimAC and MimG were fused with His-tags and purified using affinity chromatography in a homogenous state on SDS-PAGE. Blue native PAGE demonstrated that the quaternary structure of MimG was almost identical to that of chaperonin GroEL, indicating that its function was also similar to GroEL. Size-exclusion chromatography and ICP-AES analysis demonstrated that MimAC was assembled in the dimer of two sort of subunits and exhibited two iron atoms and at least one zinc atom per two subunits. This result indicated that MimAC possessed a dinuclear iron center, similar to other soluble diiron monooxygenase hydroxylases.</p>

scholarly journals Isolation and biochemical characterization of the α- and β-subunits of glycoprotein IIb of human platelet plasma membrane

1986 ◽  
Vol 240 (1) ◽  
pp. 155-161 ◽  
Author(s):  
J J Calvete ◽  
J González-Rodríguez
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Biochemical Characterization ◽  
Human Platelet ◽  
Size Exclusion ◽  
Beta Subunits ◽  
Exclusion Chromatography ◽  
Stepwise Reduction ◽  
Acidic Nature

The alpha- and beta-subunits of glycoprotein IIb (GPIIb) of human platelet plasma membrane were isolated in fully reduced, partially reduced and alkylated, and fully alkylated forms, by size-exclusion chromatography after reduction of pure GPIIb. The sugar moiety of GPIIb alpha accounts for 16.4% of its total weight, whereas that of GPIIb beta accounts for only 10.2%. The molar percentages (per 100 mol of total amino acids) of neuraminic acid and galactose in the alpha-subunit more than double those in the beta-subunit, whereas galactosamine is present only in GPIIb alpha. From the amino acid and sugar compositions the acidic nature of both subunits was confirmed. The Mr values obtained, 114,000 for GPIIb alpha and 22,200 for GPIIb beta, are in very good agreement with those obtained by physical methods. We found by stepwise reduction of pure GPIIb with dithioerythritol that GPIIb alpha and GPIIb beta are joined by a single interchain disulphide bridge, while the remaining half-cystine residues participate in intrachain bonds, six in GPIIb alpha and one in GPIIb beta, the intersubunit disulphide bond being that reduced first. Neither of the two subunits is liberated from isolated plasma membranes when this GPIIb interchain bond is reduced in isolated membranes.

scholarly journals New isolation procedure and further biochemical characterization of glycoproteins IIb and IIIa from human platelet plasma membrane

1986 ◽  
Vol 240 (1) ◽  
pp. 147-153 ◽  
Author(s):  
M T Eirín ◽  
J J Calvete ◽  
J González-Rodríguez
Keyword(s):  
Plasma Membrane ◽  
High Performance ◽  
Tertiary Structure ◽  
Biochemical Characterization ◽  
Human Platelet ◽  
Sugar Content ◽  
Size Exclusion ◽  
Apparent Lack ◽  
Weight Percentage ◽  
Triton X

We describe a new procedure for isolation of glycoproteins IIb (GPIIb) and IIIa (GPIIIa) from human platelet plasma membrane with high yields (2.7 mg of GPIIb and 3.3 mg of GPIIIa per 100 mg of starting platelet membrane proteins), equivalent to a recovery of 35% and 55% respectively of the total GPIIb and GPIIIa of the membrane. The procedure involves Triton X-100 differential extraction of platelet membranes, SDS solubilization of the 4%-Triton X-100 supernatant, zonal centrifugation in a sucrose density gradient, and preparative high-performance size-exclusion chromatography. The weight percentage of sugar is 15.7% for GPIIb and 12.5% for GPIIIa. Neuraminic acid is present in both glycoproteins, representing 30% and 15% respectively of the total sugar weight of GPIIb and GPIIIa. Mannose, galactose and glucosamine account for 45%, 13% and 28% respectively of the sugars of GPIIIa, whereas galactosamine was not detected. Mannose, galactose, glucosamine and galactosamine represent 17%, 21%, 24% and 10% respectively of the sugar content of GPIIb. The molar percentages of half-cystine and methionine are 4-fold and 2-fold higher respectively in GPIIIa than in GPIIb. From the amino acid and sugar compositions we confirmed the acidic nature of both glycoproteins. The Mr values obtained, 136,500 for GPIIb and 91,500 for GPIIIa, are in very good agreement with those obtained by physical methods. The apparent lack of free thiol groups in both glycoproteins indicates that the tertiary structure of GPIIIa is maintained by 21 intrachain disulphide bonds, and that there are eight intrachain and interchain disulphide groups in GPIIb.

scholarly journals Purification and biochemical characterization of native ERp29 from rat liver

2004 ◽  
Vol 383 (3) ◽  
pp. 589-597 ◽  
Author(s):  
Michael J. HUBBARD ◽  
Jonathan E. MANGUM ◽  
Nicola J. McHUGH
Keyword(s):  
Calcium Binding ◽  
Biochemical Characterization ◽  
Biological Significance ◽  
Size Exclusion ◽  
Secretory Proteins ◽  
Unexpected Finding ◽  
Animal Cells ◽  
Exclusion Chromatography ◽  
Helper Activity ◽  
Distinct Member

ERp29 is a recently characterized resident of the ER (endoplasmic reticulum) lumen that has broad biological significance, being expressed ubiquitously and abundantly in animal cells. As an apparent housekeeper, ERp29 is thought to be a general folding assistant for secretory proteins and to probably function as a PDI (protein disulphide isomerase)-like molecular chaperone. In the present paper, we report the first purification to homogeneity and direct functional analysis of native ERp29, which has led to the unexpected finding that ERp29 lacks PDI-like folding activities. ERp29 was purified 4800-fold in non-denaturing conditions exploiting an unusual affinity for heparin. Two additional biochemical hallmarks that will assist the classification of ERp29 homologues were identified, namely the idiosyncratic behaviours of ERp29 on size-exclusion chromatography (Mr<globular homodimer) and SDS/PAGE (Mr>monomeric mass). In contrast with PDI and parallel-purified co-residents (calreticulin, ERp60), native ERp29 lacked classical chaperone, disulphide reductase and isomerase, and calcium-binding activities. In the chaperone assays, ERp29 neither protected substrate proteins against thermal aggregation nor interacted stably with chemically denatured proteins as detected by cross-linking. ERp29 also did not exhibit helper activity toward calreticulin (chaperone) or PDI and ERp60 (disulphide reductase). By refuting long-standing predictions about chaperone activity, these results expose ERp29 as a functionally distinct member of the ER machinery and prompt a revised hypothesis that ERp29 acts as a non-classical folding assistant. The native preparation and biochemical hallmarks established here provide a useful foundation for ongoing efforts to resolve the functional orphan status of ERp29.

scholarly journals Structural analyses of the Group A flavin-dependent monooxygenase PieE reveal a sliding FAD cofactor conformation bridging OUT and IN conformations

2020 ◽  
Vol 295 (14) ◽  
pp. 4709-4722
Author(s):  
Mahder S. Manenda ◽  
Marie-Ève Picard ◽  
Liping Zhang ◽  
Normand Cyr ◽  
Xiaojun Zhu ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Underlying Mechanism ◽  
Size Exclusion ◽  
Futile Cycle ◽  
Oxygen Oxygen ◽  
Exclusion Chromatography ◽  
Group A ◽  
Nadph Oxidation ◽  
Chemical Events ◽  
First Time

Group A flavin-dependent monooxygenases catalyze the cleavage of the oxygen–oxygen bond of dioxygen, followed by the incorporation of one oxygen atom into the substrate molecule with the aid of NADPH and FAD. These flavoenzymes play an important role in many biological processes, and their most distinct structural feature is the choreographed motions of flavin, which typically adopts two distinct conformations (OUT and IN) to fulfill its function. Notably, these enzymes seem to have evolved a delicate control system to avoid the futile cycle of NADPH oxidation and FAD reduction in the absence of substrate, but the molecular basis of this system remains elusive. Using protein crystallography, size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), and small-angle X-ray scattering (SEC-SAXS) and activity assay, we report here a structural and biochemical characterization of PieE, a member of the Group A flavin-dependent monooxygenases involved in the biosynthesis of the antibiotic piericidin A1. This analysis revealed that PieE forms a unique hexamer. Moreover, we found, to the best of our knowledge for the first time, that in addition to the classical OUT and IN conformations, FAD possesses a “sliding” conformation that exists in between the OUT and IN conformations. This observation sheds light on the underlying mechanism of how the signal of substrate binding is transmitted to the FAD-binding site to efficiently initiate NADPH binding and FAD reduction. Our findings bridge a gap currently missing in the orchestrated order of chemical events catalyzed by this important class of enzymes.

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