Identification of amino acids involved in the hydrolytic activity of lipase LipBL from Marinobacter lipolyticus

The Angiotensin-I-converting enzyme (ACE) is a peptidase with a significant role in the regulation of blood pressure. Within this work, a systematic review on the enzymatic preparation of Angiotensin-I-Converting Enzyme inhibitory (ACEi) peptides is presented. The systematic review is conducted by following PRISMA guidelines. Soybeans and velvet beans are known to have high protein contents that make them suitable as sources of parent proteins for the production of ACEi peptides. Endopeptidase is commonly used in the preparation of soybean-based ACEi peptides, whereas for velvet bean, a combination of both endo- and exopeptidase is frequently used. Soybean glycinin is the preferred substrate for the preparation of ACEi peptides. It contains proline as one of its major amino acids, which exhibits a potent significance in inhibiting ACE. The best enzymatic treatments for producing ACEi peptides from soybean are as follows: proteolytic activity by Protease P (Amano-P from Aspergillus sp.), a temperature of 37 °C, a reaction time of 18 h, pH 8.2, and an E/S ratio of 2%. On the other hand, the best enzymatic conditions for producing peptide hydrolysates with high ACEi activity are through sequential hydrolytic activity by the combination of pepsin-pancreatic, an E/S ratio for each enzyme is 10%, the temperature and reaction time for each proteolysis are 37 °C and 0.74 h, respectively, pH for pepsin is 2.0, whereas for pancreatin it is 7.0. As an underutilized pulse, the studies on the enzymatic hydrolysis of velvet bean proteins in producing ACEi peptides are limited. Conclusively, the activity of soybean-based ACEi peptides is found to depend on their molecular sizes, the amino acid residues, and positions. Hydrophobic amino acids with nonpolar side chains, positively charged, branched, and cyclic or aromatic residues are generally preferred for ACEi peptides.

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Antibody Mapping of the Linear Epitopes of CMY-2 and SHV-1 β-Lactamases

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.10.3980-3988.2004 ◽

2004 ◽

Vol 48 (10) ◽

pp. 3980-3988 ◽

Cited By ~ 15

Author(s):

Andrea M. Hujer ◽

Christopher R. Bethel ◽

Robert A. Bonomo

Keyword(s):

Amino Acids ◽

Polyclonal Antibodies ◽

Synthesis Method ◽

Hydrolytic Activity ◽

Spot Synthesis ◽

Antibody Recognition ◽

Molar Excess ◽

Omega Loop ◽

Direct Binding ◽

Linear Epitopes

ABSTRACT Knowledge of the amino acids that define recognition of anti-β-lactamase antibodies is critical to the interpretation of sensitivity and specificity of these antibodies when they are used in a clinical or research setting. To this end, we mapped the epitopes of the CMY-2 and SHV-1 β-lactamases by using the SPOT synthesis method. Eight linear epitopes in SHV-1 and seven linear epitopes in CMY-2 were identified by using anti-SHV-1 and anti-CMY-2 polyclonal antibodies, respectively. The epitopes of SHV-1 were mapped to amino acids at the Ambler positions ABL 28 to 38, 42 to 54, 88 to 100, 102 to 114, 170 to 182, 186 to 194, 202 to 210, and 276 to 288. In the epitope spanning amino acids 102 to 114, alanine and X-Scan analysis demonstrated that D104, Y105, P107, and S109 are essential residues for antibody recognition. In the epitope containing amino acids 170 to 182, N170, L173, P174, G175, and D176 were immunodominant. In CMY-2 β-lactamase, amino acids 4 to 16, 70 to 79, 211 to 223, 274 to 286, 289 to 298, 322 to 334, and 343 to 358 of the mature enzyme defined the major linear epitopes. A detailed analysis of the recognition sites that are located in an area analogous to the omega loop of class A β-lactamases (V211 to V223) showed that the amino acids Q215 to E219 are important in antibody binding. Incubation of CMY-2 β-lactamase with a 10-fold molar excess of anti-CMY-2 antibody for 60 min resulted in greater than 80% inhibition of nitrocefin hydrolysis. A 10-fold molar excess of anti-SHV-1 antibody reduced the activity of SHV-1 by 69%. Analysis of the CMY-2 and SHV-1 structures suggest that this reduction of hydrolytic activity may be due in part to the direct binding of antibodies to the omega loop, thereby hindering access of substrate to the active site.

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Estimation of the Phospholipase A2 Selectivity on POPC/POPG Membranes Using the Interaction Map

Biochemistry (Moscow) Supplement Series A Membrane and Cell Biology ◽

10.1134/s1990747821050032 ◽

2021 ◽

Author(s):

A. S. Alekseeva ◽

P. E. Volynsky ◽

I. A. Boldyrev

Keyword(s):

Amino Acids ◽

Phospholipase A2 ◽

Membrane Surface ◽

Membrane Binding ◽

Hydrolytic Activity ◽

Mapping Method ◽

Lipid Mobility ◽

Lipid Protein Interaction ◽

Interaction Map ◽

Lipid Environment

Abstract The regulation of the activity and selectivity of phospholipase A2 (PLA2), which is capable of cleaving fatty acid in the second position (sn-2) of the phospholipid, is carried out through the membrane-binding and catalytic sites of the enzyme. For hydrolytic activity, PLA2 must first bind to the phospholipid membrane, and the binding efficiency depends on the composition of the membrane. The membrane-binding site of PLA2 is formed by several tens of amino acids and its composition differs from enzyme to enzyme; hydrophobic and positively charged amino acids play a key role in the interaction. In this work, we investigated the interaction of PLA2 from bee venom with phospholipid bilayers of palmitoyl oleoylphosphatidylcholine (POPC) containing different amounts of palmitoyloleoylphosphatidylglycerol (POPG). On the basis of the measurements of the protein intrinsic fluorescence and the anisotropy of the fluorescence of the lipid probe we propose the construction of lipid–protein interaction maps, which reflect both the efficiency of protein binding and changes in the structure of the membrane. These changes cause alterations in the fluorescence anisotropy of the label, which in turn is a measure of the mobility of the lipid environment of the fluorescent probe. Analysis of interaction maps showed that there is a relationship between lipid mobility and enzyme binding efficiency: the optimum interaction of PLA2 with membranes from a POPC/POPG mixture lies in the region of the highest lipid mobility, and not in the region of the highest negative charge. This dependence complements the existing understanding of the process of recognition of the membrane surface by the enzyme and the selection of lipids by the enzyme already bound to the membrane. The proposed mapping method can be extended to other membrane-active proteins.

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Molecular and Physiological Role of the Trehalose-Hydrolyzing α-Glucosidase from Thermus thermophilus HB27

Journal of Bacteriology ◽

10.1128/jb.01794-07 ◽

2008 ◽

Vol 190 (7) ◽

pp. 2298-2305 ◽

Cited By ~ 11

Author(s):

Susana Alarico ◽

Milton S. da Costa ◽

Nuno Empadinhas

Keyword(s):

Amino Acids ◽

Minimal Medium ◽

Thermus Thermophilus ◽

Physiological Role ◽

Hydrolytic Activity ◽

Recombinant Enzyme ◽

New Feature ◽

Hydrolysis Of ◽

Reciprocal Replacement

ABSTRACT Trehalose supports the growth of Thermus thermophilus strain HB27, but the absence of obvious genes for the hydrolysis of this disaccharide in the genome led us to search for enzymes for such a purpose. We expressed a putative α-glucosidase gene (TTC0107), characterized the recombinant enzyme, and found that the preferred substrate was α,α-1,1-trehalose, a new feature among α-glucosidases. The enzyme could also hydrolyze the disaccharides kojibiose and sucrose (α-1,2 linkage), nigerose and turanose (α-1,3), leucrose (α-1,5), isomaltose and palatinose (α-1,6), and maltose (α-1,4) to a lesser extent. Trehalose was not, however, a substrate for the highly homologous α-glucosidase from T. thermophilus strain GK24. The reciprocal replacement of a peptide containing eight amino acids in the α-glucosidases from strains HB27 (LGEHNLPP) and GK24 (EPTAYHTL) reduced the ability of the former to hydrolyze trehalose and provided trehalose-hydrolytic activity to the latter, showing that LGEHNLPP is necessary for trehalose recognition. Furthermore, disruption of the α-glucosidase gene significantly affected the growth of T. thermophilus HB27 in minimal medium supplemented with trehalose, isomaltose, sucrose, or palatinose, to a lesser extent with maltose, but not with cellobiose (not a substrate for the α-glucosidase), indicating that the α-glucosidase is important for the assimilation of those four disaccharides but that it is also implicated in maltose catabolism.

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Catalytic Hydrolysis of Phosphate Monoester by Supramolecular Complexes Formed by the Self-Assembly of a Hydrophobic Bis(Zn2+-cyclen) Complex, Copper, and Barbital Units That Are Functionalized with Amino Acids in a Two-Phase Solvent System

Micromachines ◽

10.3390/mi10070452 ◽

2019 ◽

Vol 10 (7) ◽

pp. 452

Author(s):

Yuya Miyazawa ◽

Akib Bin Rahman ◽

Yutaka Saga ◽

Hiroki Imafuku ◽

Yosuke Hisamatsu ◽

...

Keyword(s):

Aqueous Solution ◽

Amino Acids ◽

Active Site ◽

Self Assembly ◽

Hydrolytic Activity ◽

Solvent System ◽

Supramolecular Complexes ◽

Artificial System ◽

Two Phase ◽

Hydrolysis Of

We previously reported on the preparation of supramolecular complexes by the 2:2:2 assembly of a dinuclear Zn2+-cyclen (cyclen = 1,4,7,10-tetraazacyclododecane) complex having a 2,2′-bipyridyl linker equipped with 0~2 long alkyl chains (Zn2L1~Zn2L3), 5,5-diethylbarbituric acid (Bar) derivatives, and a copper(II) ion (Cu2+) in aqueous solution and two-phase solvent systems and their phosphatase activities for the hydrolysis of mono(4-nitrophenyl) phosphate (MNP). These supermolecules contain Cu2(μ-OH)2 core that mimics the active site of alkaline phosphatase (AP), and one of the ethyl groups of the barbital moiety is located in close proximity to the Cu2(μ-OH)2 core. The generally accepted knowledge that the amino acids around the metal center in the active site of AP play important roles in its hydrolytic activity inspired us to modify the side chain of Bar with various functional groups in an attempt to mimic the active site of AP in the artificial system, especially in two-phase solvent system. In this paper, we report on the design and synthesis of new supramolecular complexes that are prepared by the combined use of bis(Zn2+-cyclen) complexes (Zn2L1, Zn2L2, and Zn2L3), Cu2+, and Bar derivatives containing amino acid residues. We present successful formation of these artificial AP mimics with respect to the kinetics of the MNP hydrolysis obeying Michaelis–Menten scheme in aqueous solution and a two-phase solvent system and to the mode of the product inhibition by inorganic phosphate.

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Cloning, Sequencing, and Characterization of a Heat- and Alkali-Stable Type I Pullulanase from Anaerobranca gottschalkii

Applied and Environmental Microbiology ◽

10.1128/aem.70.6.3407-3416.2004 ◽

2004 ◽

Vol 70 (6) ◽

pp. 3407-3416 ◽

Cited By ~ 55

Author(s):

Costanzo Bertoldo ◽

Martin Armbrecht ◽

Fiona Becker ◽

Thomas SchÃ¯Â¿Â½fer ◽

Garabed Antranikian ◽

...

Keyword(s):

Amino Acids ◽

Molecular Mass ◽

Signal Peptide ◽

Thermotoga Maritima ◽

Sodium Dodecyl ◽

Anaerobic Bacteria ◽

Hydrolytic Activity ◽

Homologous Gene ◽

Type I ◽

E Coli

ABSTRACT The gene encoding a type I pullulanase was identified from the genome sequence of the anaerobic thermoalkaliphilic bacterium Anaerobranca gottschalkii. In addition, the homologous gene was isolated from a gene library of Anaerobranca horikoshii and sequenced. The proteins encoded by these two genes showed 39% amino acid sequence identity to the pullulanases from the thermophilic anaerobic bacteria Fervidobacterium pennivorans and Thermotoga maritima. The pullulanase gene from A. gottschalkii (encoding 865 amino acids with a predicted molecular mass of 98 kDa) was cloned and expressed in Escherichia coli strain BL21(DE3) so that the protein did not have the signal peptide. Accordingly, the molecular mass of the purified recombinant pullulanase (rPulAg) was 96 kDa. Pullulan hydrolysis activity was optimal at pH 8.0 and 70Ã¯Â¿Â½C, and under these physicochemical conditions the half-life of rPulAg was 22 h. By using an alternative expression strategy in E. coli Tuner(DE3)(pLysS), the pullulanase gene from A. gottschalkii, including its signal peptide-encoding sequence, was cloned. In this case, the purified recombinant enzyme was a truncated 70-kDa form (rPulAg′). The N-terminal sequence of purified rPulAg′ was found 252 amino acids downstream from the start site, presumably indicating that there was alternative translation initiation or N-terminal protease cleavage by E. coli. Interestingly, most of the physicochemical properties of rPulAg′ were identical to those of rPulAg. Both enzymes degraded pullulan via an endo-type mechanism, yielding maltotriose as the final product, and hydrolytic activity was also detected with amylopectin, starch, β-limited dextrins, and glycogen but not with amylose. This substrate specificity is typical of type I pullulanases. rPulAg was inhibited by cyclodextrins, whereas addition of mono- or bivalent cations did not have a stimulating effect. In addition, rPulAg′ was stable in the presence of 0.5% sodium dodecyl sulfate, 20% Tween, and 50% Triton X-100. The pullulanase from A. gottschalkii is the first thermoalkalistable type I pullulanase that has been described.

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Targeting of Porcine Pancreatic Phospholipase A2 to Human Platelets: Introduction of an RGD Sequence by Genetic Engineering

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649894 ◽

1995 ◽

Vol 74 (04) ◽

pp. 1138-1144 ◽

Cited By ~ 4

Author(s):

A C A P A Bekkers ◽

H van der Vuurst ◽

G van Willigen ◽

J W N Akkerman ◽

H M Verheij

Keyword(s):

Amino Acids ◽

Phospholipase A2 ◽

Hydrolytic Activity ◽

Human Platelets ◽

Rgd Sequence ◽

Pancreatic Phospholipase ◽

Pancreatic Phospholipase A2 ◽

Activated Platelets ◽

C Terminus ◽

Platelet Receptor

SummaryThe possibility to induce specific disruption of activated platelets by binding of porcine pancreatic phospholipase A2 (PLA2) was tested by constructing a set of PLA2-mutants containing an Arg-Gly-Asp (RGD) sequence. One mutant was made with RGD as part of a surface-exposed loop (RGDloop). Four mutants were made with RGD as part of a C-terminal extension: one with RGD directly coupled to the C-terminus (RGDc) and three mutants (CRSx) with x = 22,42 and 82 hydrophylic non-charged amino acids between RGD and the enzyme. All mutants retained 20-80% activity of native PLA2 and showed little binding to resting platelets. The binding of the native enzyme and RGDloop was not increased following stimulation. In contrast, the mutants RGDc and CRSx showed stimulation-dependent binding to the platelet receptor GPIIb/IIIa, since GRGDS-peptide and a monoclonal antibody against the complex interfered with binding. In α-thrombin-stimulated platelets, CRS42 and CRS82 induced about 5% hydrolysis of [3H]-arachidonic acid-labeled phospholipids. Stimulation with a combination of a-thrombin and collagen (known to expose phosphatidylserine) increased hydrolysis to 11%. Despite the membrane disruption, the cells did not leak lactate dehydrogenase. We conclude that PLA2 can be targeted to activated platelets by introducing RGD in a C-terminal extension with a minimum distance (42 amino acids) between RGD and the enzyme. However, more hydrolytic activity is required to eliminate activated platelets among a suspension of resting platelets and other blood cells.

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Identification of IspC, an 86-Kilodalton Protein Target of Humoral Immune Response to Infection with Listeria monocytogenes Serotype 4b, as a Novel Surface Autolysin

Journal of Bacteriology ◽

10.1128/jb.01375-06 ◽

2006 ◽

Vol 189 (5) ◽

pp. 2046-2054 ◽

Cited By ~ 11

Author(s):

Linru Wang ◽

Min Lin

Keyword(s):

Amino Acids ◽

Immune Response ◽

Cell Wall ◽

Listeria Monocytogenes ◽

Humoral Immune Response ◽

Catalytic Domain ◽

Hydrolytic Activity ◽

Ph Optimum ◽

Humoral Immune ◽

Serotype 4B

ABSTRACT We identified and biochemically characterized a novel surface-localized autolysin from Listeria monocytogenes serotype 4b, an 86-kDa protein consisting of 774 amino acids and known from our previous studies as the target (designated IspC) of the humoral immune response to listerial infection. Recombinant IspC, expressed in Escherichia coli, was purified and used to raise specific rabbit polyclonal antibodies for protein characterization. The native IspC was detected in all growth phases at a relatively stable low level during a 22-h in vitro culture, although its gene was transiently transcribed only in the early exponential growth phase. This and our previous findings suggest that IspC is upregulated in vivo during infection. The protein was unevenly distributed in clusters on the cell surface, as shown by immunofluorescence and immunogold electron microscopy. The recombinant IspC was capable of hydrolyzing not only the cell walls of the gram-positive bacterium Micrococcus lysodeikticus and the gram-negative bacterium E. coli but also that of the IspC-producing strain of L. monocytogenes serotype 4b, indicating that it was an autolysin. The IspC autolysin exhibited peptidoglycan hydrolase activity over a broad pH range of between 3 and 9, with a pH optimum of 7.5 to 9. Analysis of various truncated forms of IspC for cell wall-hydrolyzing or -binding activity has defined two separate functional domains: the N-terminal catalytic domain (amino acids [aa] 1 to 197) responsible for the hydrolytic activity and the C-terminal domain (aa 198 to 774) made up of seven GW modules responsible for anchoring the protein to the cell wall. In contrast to the full-length IspC, the N-terminal catalytic domain showed hydrolytic activity at acidic pHs, with a pH optimum of between 4 and 6 and negligible activity at alkaline pHs. This suggests that the cell wall binding domain may be of importance in modulating the activity of the N-terminal hydrolase domain. Elucidation of the biochemical properties of IspC may have provided new insights into its biological function(s) and its role in pathogenesis.

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Homochirality as the Signature of Extra-Terrestrial Life

International Astronomical Union Colloquium ◽

10.1017/s0252921100015037 ◽

1997 ◽

Vol 161 ◽

pp. 505-510

Author(s):

Alexandra J. MacDermott ◽

Laurence D. Barron ◽

Andrè Brack ◽

Thomas Buhse ◽

John R. Cronin ◽

...

Keyword(s):

Amino Acids ◽

Protein Synthesis ◽

Optical Rotation ◽

Physical Origin ◽

Natural Choice ◽

Rosetta Mission ◽

Terrestrial Life ◽

Subsurface Layers ◽

Solar System Bodies ◽

Origin Of Homochirality

AbstractThe most characteristic hallmark of life is its homochirality: all biomolecules are usually of one hand, e.g. on Earth life uses only L-amino acids for protein synthesis and not their D mirror images. We therefore suggest that a search for extra-terrestrial life can be approached as a Search for Extra- Terrestrial Homochirality (SETH). The natural choice for a SETH instrument is optical rotation, and we describe a novel miniaturized space polarimeter, called the SETH Cigar, which could be used to detect optical rotation as the homochiral signature of life on other planets. Moving parts are avoided by replacing the normal rotating polarizer by multiple fixed polarizers at different angles as in the eye of the bee. We believe that homochirality may be found in the subsurface layers on Mars as a relic of extinct life, and on other solar system bodies as a sign of advanced pre-biotic chemistry. We discuss the chiral GC-MS planned for the Roland lander of the Rosetta mission to a comet and conclude with theories of the physical origin of homochirality.

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Hydrogen Cyanide Polymers from the Impact of Comet P/Shoemaker-Levy 9 on Jupiter

International Astronomical Union Colloquium ◽

10.1017/s025292110001469x ◽

1997 ◽

Vol 161 ◽

pp. 179-187

Author(s):

Clifford N. Matthews ◽

Rose A. Pesce-Rodriguez ◽

Shirley A. Liebman

Keyword(s):

Amino Acids ◽

Solar System ◽

Hydrogen Cyanide ◽

Nitrogen Heterocycles ◽

Laboratory Studies ◽

Heterogeneous Solids ◽

The Many ◽

Comet Halley ◽

The Impact ◽

By Products

AbstractHydrogen cyanide polymers – heterogeneous solids ranging in color from yellow to orange to brown to black – may be among the organic macromolecules most readily formed within the Solar System. The non-volatile black crust of comet Halley, for example, as well as the extensive orangebrown streaks in the atmosphere of Jupiter, might consist largely of such polymers synthesized from HCN formed by photolysis of methane and ammonia, the color observed depending on the concentration of HCN involved. Laboratory studies of these ubiquitous compounds point to the presence of polyamidine structures synthesized directly from hydrogen cyanide. These would be converted by water to polypeptides which can be further hydrolyzed to α-amino acids. Black polymers and multimers with conjugated ladder structures derived from HCN could also be formed and might well be the source of the many nitrogen heterocycles, adenine included, observed after pyrolysis. The dark brown color arising from the impacts of comet P/Shoemaker-Levy 9 on Jupiter might therefore be mainly caused by the presence of HCN polymers, whether originally present, deposited by the impactor or synthesized directly from HCN. Spectroscopic detection of these predicted macromolecules and their hydrolytic and pyrolytic by-products would strengthen significantly the hypothesis that cyanide polymerization is a preferred pathway for prebiotic and extraterrestrial chemistry.

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