{"title":"A novel alcohol dehydrogenase in the hyperthermophilic crenarchaeon <i>Hyperthermus butylicus</i>.","authors":"Ching Tse, Kesen Ma","doi":"10.1002/mlf2.12126","DOIUrl":null,"url":null,"abstract":"<p><p><i>Hyperthermus butylicus</i> is a hyperthermophilic crenarchaeon that produces 1-butanol as an end product. A thermostable alcohol dehydrogenase (ADH) must be present in <i>H. butylicus</i> to act as the key enzyme responsible for this production; however, the gene that encodes the ADH has not yet been identified. A novel ADH, HbADH2, was purified from a cell-free extract of <i>H. butylicus</i>, and its characteristics were determined. The gene that encodes HbADH2 was demonstrated to be <i>HBUT_RS04850</i> and annotated as a hypothetical protein in <i>H. butylicus</i>. HbADH2 was found to be a primary-secondary ADH capable of using a wide range of substrates, including butyraldehyde and butanol. Butyraldehyde had the highest specificity constant, calculated as <i>k</i> <sub>c</sub> <sub>at</sub>/<i>K</i> <sub>m</sub>, with <i>k</i> <sub>cat</sub> and apparent <i>K</i> <sub>m</sub> values of 8.00 ± 0.22 s<sup>-1</sup> and 0.59 ± 0.07 mM, respectively. The apparent <i>K</i> <sub>m</sub> values for other substrates, including ethanol, 1-propanol, 2-propanol, butanol, acetaldehyde, propanal, and acetone, were 4.36 ± 0.42, 4.69 ± 0.41, 3.74 ± 0.46, 2.44 ± 0.30, 1.27 ± 0.18, 1.55 ± 0.20, and 0.68 ± 0.04 mM, respectively. The optimal pH values for catalyzing aldehyde reduction and alcohol oxidation were 6.0 and 9.0, respectively, while the optimal temperature was higher than 90°C due to the increase in enzymatic activity from 60°C to 90°C. Based on its substrate specificity, enzyme kinetics, and thermostability, HbADH2 may be the ADH that catalyzes the production of 1-butanol in <i>H. butylicus</i>. The putative conserved motif sites for NAD(P)<sup>+</sup> and iron binding were identified by aligning HbADH2 with previously characterized Fe-containing ADHs.</p>","PeriodicalId":94145,"journal":{"name":"mLife","volume":null,"pages":null},"PeriodicalIF":4.5000,"publicationDate":"2024-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11211662/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"mLife","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1002/mlf2.12126","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/6/1 0:00:00","PubModel":"eCollection","JCR":"Q1","JCRName":"MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Hyperthermus butylicus is a hyperthermophilic crenarchaeon that produces 1-butanol as an end product. A thermostable alcohol dehydrogenase (ADH) must be present in H. butylicus to act as the key enzyme responsible for this production; however, the gene that encodes the ADH has not yet been identified. A novel ADH, HbADH2, was purified from a cell-free extract of H. butylicus, and its characteristics were determined. The gene that encodes HbADH2 was demonstrated to be HBUT_RS04850 and annotated as a hypothetical protein in H. butylicus. HbADH2 was found to be a primary-secondary ADH capable of using a wide range of substrates, including butyraldehyde and butanol. Butyraldehyde had the highest specificity constant, calculated as kcat/Km, with kcat and apparent Km values of 8.00 ± 0.22 s-1 and 0.59 ± 0.07 mM, respectively. The apparent Km values for other substrates, including ethanol, 1-propanol, 2-propanol, butanol, acetaldehyde, propanal, and acetone, were 4.36 ± 0.42, 4.69 ± 0.41, 3.74 ± 0.46, 2.44 ± 0.30, 1.27 ± 0.18, 1.55 ± 0.20, and 0.68 ± 0.04 mM, respectively. The optimal pH values for catalyzing aldehyde reduction and alcohol oxidation were 6.0 and 9.0, respectively, while the optimal temperature was higher than 90°C due to the increase in enzymatic activity from 60°C to 90°C. Based on its substrate specificity, enzyme kinetics, and thermostability, HbADH2 may be the ADH that catalyzes the production of 1-butanol in H. butylicus. The putative conserved motif sites for NAD(P)+ and iron binding were identified by aligning HbADH2 with previously characterized Fe-containing ADHs.