Catherine O. Esuola , Olubukola O. Babalola , Thomas Heine , Ringo Schwabe , Micheal Schlömann , Dirk Tischler (Dr.)
{"title":"Identification and characterization of a FAD-dependent putrescine N-hydroxylase (GorA) from Gordonia rubripertincta CWB2","authors":"Catherine O. Esuola , Olubukola O. Babalola , Thomas Heine , Ringo Schwabe , Micheal Schlömann , Dirk Tischler (Dr.)","doi":"10.1016/j.molcatb.2016.08.003","DOIUrl":null,"url":null,"abstract":"<div><p>A putrescine <em>N</em>-hydroxylase from <em>Gordonia rubripertincta</em> CWB2 (GorA), a microbial <em>N</em>-hydroxylating monooxygenase (NMO), specific for a range of diamines (putrescine<!--> <!-->><!--> <!-->cadaverine<!--> <!-->><!--> <!-->hexamethylenediamine) was identified. This NMO clustered together with some known but yet to be characterized diamine NMOs which are RhbE, from <em>Sinorhizobium meliloti</em> 1021; AlcA, from <em>Bordetella bronchiseptica</em> RB50, and DesB, from <em>Streptomyces scabiei</em> 87-22. It comprises 459 amino acids in length and has approximately a molecular weight of 51.4<!--> <!-->kDa. It has been successfully cloned, overexpressed, and purified as a soluble flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) dependent His<sub>10</sub>-tagged protein using <em>Escherichia coli</em> as the cloning and expression host and pET16bP as vector. The NAD(P)H oxidation assay and a hydroxylation assay were used to assess its biochemical properties. The pH optimum is between the range of 7.0–8.0 in a potassium phosphate buffer. 1,4-diaminobutane (putrescine) was the best substrate concerning GorA activity. With the NADPH oxidation assay, the kinetic parameters of this enzyme showed an apparent <em>K</em><sub>m</sub> and <em>k</em><sub>cat</sub> of 361.6<!--> <!-->±<!--> <!-->0.1<!--> <!-->μM and 0.266<!--> <!-->±<!--> <!-->0.011<!--> <!-->s<sup>−1</sup>, respectively, whereas the hydroxylation assay showed GorA with an apparent <em>K<sub>m</sub></em> and <em>k</em><sub>cat</sub> of 737.1<!--> <!-->±<!--> <!-->0.1<!--> <!-->μM and 0.210<!--> <!-->±<!--> <!-->0.001<!--> <!-->s<sup>−1</sup>. These activity data were obtained of kinetic experiments from fixing FAD and NADPH and varying the concentration of 1,4-diaminobutane. Thus this is the first diamine <em>N</em>-hydroxylating monooxygenase characterized with a physiological role in siderophore biosynthesis.</p></div>","PeriodicalId":16416,"journal":{"name":"Journal of Molecular Catalysis B-enzymatic","volume":"134 ","pages":"Pages 378-389"},"PeriodicalIF":0.0000,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.molcatb.2016.08.003","citationCount":"25","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Molecular Catalysis B-enzymatic","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1381117716301473","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"Chemical Engineering","Score":null,"Total":0}
引用次数: 25
Abstract
A putrescine N-hydroxylase from Gordonia rubripertincta CWB2 (GorA), a microbial N-hydroxylating monooxygenase (NMO), specific for a range of diamines (putrescine > cadaverine > hexamethylenediamine) was identified. This NMO clustered together with some known but yet to be characterized diamine NMOs which are RhbE, from Sinorhizobium meliloti 1021; AlcA, from Bordetella bronchiseptica RB50, and DesB, from Streptomyces scabiei 87-22. It comprises 459 amino acids in length and has approximately a molecular weight of 51.4 kDa. It has been successfully cloned, overexpressed, and purified as a soluble flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) dependent His10-tagged protein using Escherichia coli as the cloning and expression host and pET16bP as vector. The NAD(P)H oxidation assay and a hydroxylation assay were used to assess its biochemical properties. The pH optimum is between the range of 7.0–8.0 in a potassium phosphate buffer. 1,4-diaminobutane (putrescine) was the best substrate concerning GorA activity. With the NADPH oxidation assay, the kinetic parameters of this enzyme showed an apparent Km and kcat of 361.6 ± 0.1 μM and 0.266 ± 0.011 s−1, respectively, whereas the hydroxylation assay showed GorA with an apparent Km and kcat of 737.1 ± 0.1 μM and 0.210 ± 0.001 s−1. These activity data were obtained of kinetic experiments from fixing FAD and NADPH and varying the concentration of 1,4-diaminobutane. Thus this is the first diamine N-hydroxylating monooxygenase characterized with a physiological role in siderophore biosynthesis.
期刊介绍：
Journal of Molecular Catalysis B: Enzymatic is an international forum for researchers and product developers in the applications of whole-cell and cell-free enzymes as catalysts in organic synthesis. Emphasis is on mechanistic and synthetic aspects of the biocatalytic transformation.
Papers should report novel and significant advances in one or more of the following topics;
Applied and fundamental studies of enzymes used for biocatalysis;
Industrial applications of enzymatic processes, e.g. in fine chemical synthesis;
Chemo-, regio- and enantioselective transformations;
Screening for biocatalysts;
Integration of biocatalytic and chemical steps in organic syntheses;
Novel biocatalysts, e.g. enzymes from extremophiles and catalytic antibodies;
Enzyme immobilization and stabilization, particularly in non-conventional media;
Bioprocess engineering aspects, e.g. membrane bioreactors;
Improvement of catalytic performance of enzymes, e.g. by protein engineering or chemical modification;
Structural studies, including computer simulation, relating to substrate specificity and reaction selectivity;
Biomimetic studies related to enzymatic transformations.
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