A novel alcohol dehydrogenase in the hyperthermophilic crenarchaeon Hyperthermus butylicus.

IF 4.5 Q1 MICROBIOLOGY mLife Pub Date : 2024-06-28 eCollection Date: 2024-06-01 DOI:10.1002/mlf2.12126
Ching Tse, Kesen Ma
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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 k c at/K m, with k cat and apparent K m values of 8.00 ± 0.22 s-1 and 0.59 ± 0.07 mM, respectively. The apparent K m 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.

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高热嗜温性丁香菌(Hyperthermus butylicus)中的一种新型醇脱氢酶。
丁醇嗜热菌(Hyperthermus butylicus)是一种嗜热栗色菌,其最终产物为 1-丁醇。丁醇嗜热菌中必须存在一种可恒温的醇脱氢酶(ADH),它是产生这种产物的关键酶;然而,编码 ADH 的基因尚未确定。我们从丁酸梭菌的无细胞提取物中纯化出了一种新型 ADH--HbADH2,并确定了它的特征。编码 HbADH2 的基因被证明是 HBUT_RS04850,并被注释为丁酸杆菌中的一种假定蛋白。研究发现,HbADH2 是一种初级-次级 ADH,能够使用多种底物,包括丁醛和丁醇。丁醛的特异性常数最高,以 k c at/K m 计算,k cat 和表观 K m 值分别为 8.00 ± 0.22 s-1 和 0.59 ± 0.07 mM。其他底物(包括乙醇、1-丙醇、2-丙醇、丁醇、乙醛、丙醛和丙酮)的表观 K m 值分别为 4.36 ± 0.42、4.69 ± 0.41、3.74 ± 0.46、2.44 ± 0.30、1.27 ± 0.18、1.55 ± 0.20 和 0.68 ± 0.04 mM。催化醛还原和醇氧化的最佳 pH 值分别为 6.0 和 9.0,而最佳温度则高于 90°C,这是因为酶活性从 60°C 升高到 90°C。根据其底物特异性、酶动力学和恒温性,HbADH2 可能是催化丁酸杆菌产生 1-丁醇的 ADH。通过将 HbADH2 与先前表征的含铁 ADH 进行比对,确定了 NAD(P)+ 和铁结合的推定保守基团位点。
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