Binding and Catalytic Mechanisms of Veratryl Alcohol Oxidation by Lignin Peroxidase: A Theoretical and Experimental Study.

Lignin peroxidase (LiP) and its natural substrate veratryl alcohol (VA) play a crucial role in lignin degradation by white-rot fungi. Understanding the molecular determinants for the interaction of this enzyme with its substrates is essential in the rational design of engineered peroxidases for biotechnological application. Here, we combine computational and experimental approaches to analyze the interaction of Phanerochaete chrysosporium LiP (isoenzyme H8) with VA and its radical cation (VA•+, resulting from substrate oxidation by the enzyme). Interaction energy calculations at semiempirical quantum mechanical level (SQM) between LiP and VA/VA•+ enabled to identify those residues at the acidic environment of catalytic Trp171 involved in the main interactions. Then, a battery of variants, with single and multiple mutations at these residues (Glu168, Asp165, Glu250, Asp264, and Phe267), was generated by directed mutagenesis, and their kinetics parameters were estimated on VA and two additional substrates. The experimental results show that Glu168 and Glu250 are crucial for the binding of VA, with Glu250 also contributing to the turnover of the enzyme. The experimental results were further rationalized through new calculations of interaction energies between VA/VA•+ and LiP with each of the single mutations. Finally, the delocalization of spin density was determined with quantum mechanics/molecular mechanics calculations (QM/MM), further supporting the contribution of Glu250 to VA oxidation at Trp171.

Main plants used in traditional medicine for the treatment of snake bites n the regions of the department of Antioquia, Colombia.

ETHNOPHARMACOLOGY RELEVANCE: In Colombia, more than 4.000 ophidian accidents occur per year and due to the scarce distribution and limited availability of antivenom, the use of traditional medicine has been perpetuated in some of its rural communities, in which initially, those affected are treated by healers and shamans using medicinal plants in different ways. METHODS: Research was conducted with renowned healers or connoisseurs of plants on the ethnobotany of ophidian accidents in five different areas and their municipalities of Antioquia: Magdalena Medio (Caracolí, Puerto Berrío); Bajo Cauca (Caucasia, Zaragoza); Nordeste (San Roque, Yalí); Norte (Gómez Plata, Valdivia); Suroeste (Ciudad Bolívar, Salgar); collecting information related to experience and time of use of plants in the treatment of these poisonings, amounts used, ways of use (beverage, bathing, ointment, chupaderas, vapors), preparation types (maceration or decoction) and treatment duration. RESULTS: 71 plant species were identified and collected, 49.29% of them without previous reports as antiophidian and 38.0% employed for the same purpose in other geographical areas. The leaves (24.82%), stems (11.68%) and flowers (10.95%) were found to be the most frequently employed structures in the preparation of the extracts, which are usually prepared by decoction (83.94%), maceration (6.57%). CONCLUSIONS: In this work, specimens lacking previous ethnobotanical reports have been found, plants used by ethnic groups from other regions of Antioquia and the world to treat snake bites; and herbaceous plants whose inhibitory activity of symptoms produced by some snake venoms, has been experimentally verified by in vivo and in vitro tests.

Snakebites and ethnobotany in the Eastern region of Antioquia, Colombia--the traditional use of plants.

ETHNOPHARMACOLOGY RELEVANCE: Every year, over 4000 ophidian accidents take place in Colombia. The poor distribution and limited availability of antivenom have perpetuated the use of traditional medicine especially in rural communities. The medical care starts with the affected people receiving treatment by healers or shamans who use medicinal plants prepared in different ways. METHODS: This investigation was undertaken by conducting enquiries to the renowned healers or plants connoisseurs about the ethnobotany of the ophidian accident. In addition, this study included their experience and the time they have used plants in curing bites or stings by poisonous animals. Furthermore, it embraced the plant species used and the amount applied, the ways of their preparation (maceration or decoction), some application techniques (beverage, bathing, poultice, vapors), and duration of treatment. RESULTS: 29 species of plants were collected and identified. 82.7% of them were native, 27.5% had not been previously reported as antiophidian, and 38% had been employed for this purpose in other geographical areas. Leaves (43.59%) and stems (23.08%) were the components most frequently used in the medicinal preparation, which is usually done by maceration (51.92%) or decoction (25.00%). CONCLUSIONS: Throughout this study related to the treatment of snakebite accident, species have been found without any ethnobotanical reports. Moreover, plants being described as new specie and herbs such as Plantago major (Plantaginaceae) used in the treatment of snakebites by many ethnic groups have been found. In addition, herbaceous such as Renealmia alpinia (Zingiberaceae), whose antiophidian activity has been experimentally proven through in vivo and in vitro assays, have been discovered too.