Engineering substrate promiscuity in halophilic alcohol dehydrogenase (HvADH2) by in silico design

Cassidy, Jennifer; Bruen, Larah; Rosini, Elena; Molla, Gianluca; Pollegioni, Loredano; Paradisi, Francesca

doi:10.1371/journal.pone.0187482

Cited by 11 publications

(13 citation statements)

References 24 publications

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“…Immobilization within epoxy resin and metal organic framework material further improves the enzyme activity over a broader pH range and temperature and enhances enzyme stability in organic solvents (Alsafadi and Paradisi 2014;Carucci et al 2018). It is also possible to improve the substrate scope of HvADH2 for biocatalysis through rational in silico enzyme design (Cassidy et al 2017). H. volcanii expression systems can be used to overexpress and characterize industrially attractive enzymes from other halophiles (e.g.…”

Section: Biotechnological Potential Of H Volcaniimentioning

confidence: 99%

Haloferax volcanii for biotechnology applications: challenges, current state and perspectives

Haque

Paradisi

Allers

2019

Appl Microbiol Biotechnol

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Haloferax volcanii is an obligate halophilic archaeon with its origin in the Dead Sea. Simple laboratory culture conditions and a wide range of genetic tools have made it a model organism for studying haloarchaeal cell biology. Halophilic enzymes of potential interest to biotechnology have opened up the application of this organism in biocatalysis, bioremediation, nanobiotechnology, bioplastics and the biofuel industry. Functionally active halophilic proteins can be easily expressed in a halophilic environment, and an extensive genetic toolkit with options for regulated protein overexpression has allowed the purification of biotechnologically important enzymes from different halophiles in H. volcanii. However, corrosion mediated damage caused to stainless-steel bioreactors by high salt concentrations and a tendency to form biofilms when cultured in high volume are some of the challenges of applying H. volcanii in biotechnology. The ability to employ expressed active proteins in immobilized cells within a porous biocompatible matrix offers new avenues for exploiting H. volcanii in biotechnology. This review critically evaluates the various application potentials, challenges and toolkits available for using this extreme halophilic organism in biotechnology.

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Section: Biotechnological Potential Of H Volcaniimentioning

confidence: 99%

Haloferax volcanii for biotechnology applications: challenges, current state and perspectives

Haque

Paradisi

Allers

2019

Appl Microbiol Biotechnol

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“…While HvADH1 had some interesting activity, HvADH2 had significantly better stability, unprecedented solvent tolerance, and broader substrate scope (Timpson et al 2013). HvADH2 soon became the benchmark for halophilic alcohol dehydrogenases with extensive further investigation into further enhancing its stability by covalent immobilization (Alsafadi and Paradisi 2014), mutagenesis to probe the active site and increase activity towards non-steroidal anti-inflammatory intermediates (Cassidy 2017) as well as its effective application in the asymmetric reduction of prochiral aromatic ketones (Alsafadi 2017). Two putative ADHs were then selected from the Red Sea and expressed in Haloferax volcanii using a stirred-tank bioreactor.…”

Section: Hmadh12 (Haloarcula Marismortui) Was Heterologously Overexpressed and Purified Frommentioning

confidence: 99%

“…This strain features a double deletion of endogenous adh1 and adh2 and is our strain of choice for expression of ADHs. Expression of HwADH followed established protocols previously reported for HvADH2 (Cassidy 2017). It is worth mentioning that Hqrw_1156adh was also cloned into pRSETb to probe expression in E.coli.…”

Section: Expression and Purification Of Hwadhmentioning

confidence: 99%

Haloquadratum walsbyi Yields a Versatile, NAD+/NADP+ Dual Affinity, Thermostable, Alcohol Dehydrogenase (HwADH)

Cassidy

Paradisi

2018

Mol Biotechnol

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This study presents the first example of an alcohol dehydrogenase (ADH) from the halophilic archaeum Haloquadratum walsbyi (HwADH). A hexahistidine-tagged recombinant HwADH was heterologously overexpressed in Haloferax volcanii. HwADH was purified in one step and was found to be thermophilic with optimal activity at 65 °C. HwADH was active in the presence of 10% (v/v) organic solvent. The enzyme displayed dual cofactor specificity and a broad substrate scope, and maximum activity was detected with benzyl alcohol and 2-phenyl-1-propanol. HwADH accepted aromatic ketones, acetophenone and phenylacetone as substrates. The enzyme also accepted cyclohexanol and aromatic secondary alcohols, 1-phenylethanol and 4-phenyl-2-butanol. H. walsbyi may offer an excellent alternative to other archaeal sources to expand the toolbox of halophilic biocatalysts.

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“…Recently, an ADH from the halophilic archaeon Haloferax volcanii (HvADH2) has been identified [6] and engineered by rational design to broaden its substrate scope towards the conversion of a variety of aromatic substrates. [7] Furthermore, a bacterial β-ketoacyl-ACP reductase (FabG) from Bacillus sp. ECU0013 [8] and NADPH-dependent (S)-carbonyl reductase from C. parapsilosis ATCC 7330 [9] reduce ethyl 2-oxo-4-phenylbutanoate (EOPB) to (S)-ethyl 2-hydroxy-4-phenylbutanoate (S-EHPB).…”

Section: Introductionmentioning

confidence: 99%

“…strain, Candida parapsilosis aldehyde dehydrogenase 5 (cpADH5), and ADH from Lactobacillus kefir , which are capable of reducing ketones with bulky side chains. Recently, an ADH from the halophilic archaeon Haloferax volcanii (HvADH2) has been identified and engineered by rational design to broaden its substrate scope towards the conversion of a variety of aromatic substrates . Furthermore, a bacterial β‐ketoacyl‐ACP reductase (FabG) from Bacillus sp.…”

Section: Introductionmentioning

confidence: 99%

Reversal of Regioselectivity in Zinc‐Dependent Medium‐Chain Alcohol Dehydrogenase from Rhodococcus erythropolis toward Octanone Derivatives

et al. 2020

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The zinc-dependent medium-chain alcohol dehydrogenase from Rhodococcus erythropolis (ReADH) is one of the most versatile biocatalysts for the stereoselective reduction of ketones to chiral alcohols. Despite its known broad substrate scope, ReADH only accepts carbonyl substrates with either a methyl or an ethyl group adjacent to the carbonyl moiety; this limits its use in the synthesis of the chiral alcohols that serve as a building blocks for pharmaceuticals. Protein engineering to expand the substrate scope of ReADH toward bulky substitutions next to carbonyl group (ethyl 2-oxo-4-phenylbutyrate) opens up new routes in the synthesis of ethyl-2-hydroxy-4phenylbutanoate, an important intermediate for anti-hypertension drugs like enalaprilat and lisinopril. We have performed computer-aided engineering of ReADH toward ethyl 2-oxo-4phenylbutyrate and octanone derivatives. W296, which is located in the small binding pocket of ReADH, sterically restricts the access of ethyl 2-oxo-4-phenylbutyrate, octan-3-one or octan-4-one toward the catalytic zinc ion and thereby limits ReADH activity. Computational analysis was used to identify position W296 and site-saturation mutagenesis (SSM) yielded an improved variant W296A with a 3.6-fold improved activity toward ethyl 2-oxo-4-phenylbutyrate when compared to WT ReADH (ReADH W296A: 17.10 U/mg and ReADH WT: 4.7 U/mg). In addition, the regioselectivity of ReADH W296A is shifted toward octanone substrates. ReADH W296A has a more than 16-fold increased activity toward octan-4-one (ReADH W296A: 0.97 U/mg and ReADH WT: 0.06 U/mg) and a more than 30-fold decreased activity toward octan-2-one (ReADH W296A: 0.23 U/ mg and ReADH WT: 7.69 U/mg). Computational and experimental results revealed the role of position W296 in controlling the substrate scope and regiopreference of ReADH for a variety of carbonyl substrates.

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Engineering substrate promiscuity in halophilic alcohol dehydrogenase (HvADH2) by in silico design

Cited by 11 publications

References 24 publications

Haloferax volcanii for biotechnology applications: challenges, current state and perspectives

Haloferax volcanii for biotechnology applications: challenges, current state and perspectives

Haloquadratum walsbyi Yields a Versatile, NAD+/NADP+ Dual Affinity, Thermostable, Alcohol Dehydrogenase (HwADH)

Reversal of Regioselectivity in Zinc‐Dependent Medium‐Chain Alcohol Dehydrogenase from Rhodococcus erythropolis toward Octanone Derivatives

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