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Crystallization and preliminary X-ray diffraction studies of an L-amino-acid oxidase from Lachesis muta venom.

ABSTRACT: Snake-venom proteins form multi-component defence systems by the recruitment and rapid evolution of nonvenomous proteins and hence serve as model systems to understand the structural modifications that result in toxicity. L-Amino-acid oxidases (LAAOs) are encountered in a number of snake venoms and have been implicated in the inhibition of platelet aggregation, cytotoxicity, haemolysis, apoptosis and haemorrhage. An L-amino-acid oxidase from Lachesis muta venom has been purified and crystallized. The crystals belonged to space group P2?, with unit-cell parameters a=66.05, b=79.41, c=100.52?Å, ?=96.55°. The asymmetric unit contained two molecules and the structure has been determined and partially refined at 3.0?Å resolution.

SUBMITTER: Ullah A

PROVIDER: S-EPMC4231865 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Crystallization and preliminary X-ray diffraction studies of an L-amino-acid oxidase from Lachesis muta venom.

Ullah Anwar A Masood Rehana R Spencer Patrick Jack PJ Murakami Mário Tyago MT Arni Raghuvir Krishnaswamy RK

Acta crystallographica. Section F, Structural biology communications 20141025 Pt 11

Snake-venom proteins form multi-component defence systems by the recruitment and rapid evolution of nonvenomous proteins and hence serve as model systems to understand the structural modifications that result in toxicity. L-Amino-acid oxidases (LAAOs) are encountered in a number of snake venoms and have been implicated in the inhibition of platelet aggregation, cytotoxicity, haemolysis, apoptosis and haemorrhage. An L-amino-acid oxidase from Lachesis muta venom has been purified and crystallized ...[more]

PMID: 25372830

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Project description:BackgroundLachesis muta rhombeata is one of the venomous snakes of medical importance in Brazil whose envenoming is characterized by local and systemic effects which may produce even shock and death. Its venom is mainly comprised of serine and metalloproteinases, phospholipases A2 and bradykinin-potentiating peptides. Based on a previously reported fractionation of L. m. rhombeata venom (LmrV), we decided to perform a subproteome analysis of its major fraction and investigated a novel component present in this venom.MethodsLmrV was fractionated through molecular exclusion chromatography and the main fraction (S5) was submitted to fibrinogenolytic activity assay and fractionated by reversed-phase chromatography. The N-terminal sequences of the subfractions eluted from reversed-phase chromatography were determined by automated Edman degradation. Enzyme activity of LmrSP-4 was evaluated upon chromogenic substrates for thrombin (S-2238), plasma kallikrein (S-2302), plasmin and streptokinase-activated plasminogen (S-2251) and Factor Xa (S-2222) and upon fibrinogen. All assays were carried out in the presence or absence of possible inhibitors. The fluorescence resonance energy transfer substrate Abz-KLRSSKQ-EDDnp was used to determine the optimal conditions for LmrSP-4 activity. Molecular mass of LmrSP-4 was determined by MALDI-TOF and digested peptides after trypsin and Glu-C treatments were analyzed by high resolution MS/MS using different fragmentation modes.ResultsFraction S5 showed strong proteolytic activity upon fibrinogen. Its fractionation by reversed-phase chromatography gave rise to 6 main fractions (S5C1-S5C6). S5C1-S5C5 fractions correspond to serine proteinases whereas S5C6 represents a C-type lectin. S5C4 (named LmrSP-4) had its N-terminal determined by Edman degradation up to the 53rd amino acid residue and was chosen for characterization studies. LmrSP-4 is a fibrinogenolytic serine proteinase with high activity against S-2302, being inhibited by PMSF and benzamidine, but not by 1,10-phenantroline. In addition, this enzyme exhibited maximum activity within the pH range from neutral to basic and between 40 and 50 °C. About 68% of the LmrSP-4 primary structure was covered, and its molecular mass is 28,190 Da.ConclusionsNovel serine proteinase isoforms and a lectin were identified in LmrV. Additionally, a kallikrein-like serine proteinase that might be useful as molecular tool for investigating bradykinin-involving process was isolated and partially characterized.

Dataset Information

Crystallization and preliminary X-ray diffraction studies of an L-amino-acid oxidase from Lachesis muta venom.

Publications

Crystallization and preliminary X-ray diffraction studies of an L-amino-acid oxidase from Lachesis muta venom.

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