Structure-Guided Engineering of A Short-Chain Alcohol Dehydrogenase for Asymmetric Synthesis

Xuan Le Author

08/02/2021 Added

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This video discusses the Structure-Guided Engineering of A Short-Chain Alcohol Dehydrogenase for Asymmetric Synthesis

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[00:00:01.800]Hello, everyone. Welcome to my presentation!
[00:00:04.440]My name is Xuan Le and today I will be presenting my project on the
[00:00:08.790]Structure-Guided Engineering of a Short-Chain
[00:00:10.980]Alcohol Dehydrogenase for Asymmetric Synthesis.
[00:00:16.230]The reason
[00:00:16.830]we are interested in working on this project is because the demand for
[00:00:21.210]enantiomerically pure compounds is on the rise due to their applications in the
[00:00:25.950]chemical and pharmaceutical industries.
[00:00:28.800]These compounds can be produced using either chiral
[00:00:31.880]chemical catalyst or enzyme biocatalyst.
[00:00:35.550]This research is focused on the biocatalyst aspects due to several
[00:00:39.840]advantages over the chemical catalysts,
[00:00:42.750]such as that biocatalysts have inherently higher enantioselectivities,
[00:00:48.630]they require no toxic reagents, and they carry out
[00:00:52.830]reactions at much milder conditions.
[00:00:56.430]Specifically, this work explores an alcohol dehydrogenase that was
[00:01:00.900]cloned from Clostritium Acetobutylicum,
[00:01:04.680]which we'll refer to as CaADH. Like other alcohol
[00:01:09.540]dehydrogenases, this enzyme catalyzes,
[00:01:12.570]the conversions between alcohols and aldehydes or ketones.
[00:01:17.610]Previous works have shown that CaADH has broad substrate specificity
[00:01:22.470]and high stereochemical fidelity,
[00:01:26.370]and it can potentially be used to produce tesetaxel
[00:01:29.700]building block. Figure 1 here gives us an idea of the
[00:01:34.350]enzyme's mechanism.
[00:01:36.780]What's interesting about this enzyme is that the starting
[00:01:41.580]material does not necessarily need to be pure. As shown here,
[00:01:46.470]The starting material is actually a mixture of two stereo-isomers.
[00:01:50.780]And in the end,
[00:01:53.370]only the compounds of this specific stereo-configuration
[00:01:57.810]was obtained. In addition,
[00:02:01.770]CaADH utilizes NADPH as cofactor,
[00:02:07.110]which is recycled through this glucose dehydrogenase pathway.
[00:02:13.230]The goal here is to characterize and engineer,
[00:02:16.120]this enzyme so that its kinetic properties are improved on the substrates of
[00:02:21.000]interest.
[00:02:23.070]Some experimental procedure performed include site-directed
[00:02:27.330]mutagenesis, where a kit from New England Biolabs were used.
[00:02:31.470]And I will explain more about this kit a little bit later. In addition,
[00:02:35.730]the enzyme was purified using protein purification technique and
[00:02:40.320]characterized using enzyme assays.
[00:02:45.650]The engineering effort started with analyzing the available crystal structure of
[00:02:50.300]CaADH in complex with NADP cofactor,
[00:02:54.440]which is shown here in Figure 2A.
[00:02:56.480]Most of the protein was included in this structure,
[00:03:01.450]however,
[00:03:02.080]residues 191 through 204 were missing.
[00:03:07.270]We hypothesized that these missing residues comprise of a flexible loop
[00:03:11.950]region highlighted as red in Figure 2B,
[00:03:15.910]which plays an important role in substrate binding and turnover rates.
[00:03:21.160]In order to test this hypothesis,
[00:03:23.260]alanine scanning was conducted on these residues. Alanine
[00:03:27.200]scanning is a site-directed mutagenesis technique where alanine scanning
[00:03:31.780]libraries are used to identify the residues that play roles in the functioning
[00:03:36.550]of a protein. A library is constructed by replacing residues of
[00:03:41.410]interest with an alanine. Alanine is chosen because it is functionally
[00:03:46.390]inert yet structurally versatile. Mutants in the obtained
[00:03:50.200]library are then characterized. Mutations of key residues
[00:03:55.090]will lead to significant change in protein properties.
[00:03:59.980]We generated an alanine scanning library for residues
[00:04:03.790]191 through 204 of CaADH.
[00:04:09.460]And as I mentioned before,
[00:04:10.960]this experiment was carried out using a kit from New England Biolabs
[00:04:16.000]Figure 3 here is an overview of how the kit works.
[00:04:20.620]The first step is an exponential amplification using standard
[00:04:25.260]primers and a master mix.
[00:04:27.970]The second step involves incubation with a unique
[00:04:32.590]enzyme mix,
[00:04:33.520]which allows for rapid circularization of the PCR products and
[00:04:38.290]removal of template DNA. And lastly,
[00:04:42.010]a high-efficiency transformation into chemically competent cells is
[00:04:46.960]performed. After the mutants were generated,
[00:04:50.980]they were kinetically characterized on the five substrates of interests labelled
[00:04:55.810]1 through 5 here From Figure 4a,
[00:04:59.080]we saw that mutants V199A and
[00:05:03.700]K200A had the highest overall activities on
[00:05:08.140]substrates 1, 2, and 3.
[00:05:10.870]These two mutants were further characterized on two additional substrates,
[00:05:15.550]4 and 5. Improved activities in comparing with the wild type
[00:05:20.380]was seen.
[00:05:23.190]In conclusion, mutations of all residues to alanine had
[00:05:27.840]substantial effects on CaADH activities. For future work,
[00:05:32.880]activities of other mutants besides V199A and K200A would be
[00:05:37.770]examined on substrate 4 and 5. Future engineering of
[00:05:42.300]CaADH will combined results of alanine scanning and structural
[00:05:46.950]study. And finally,
[00:05:49.620]I would like to acknowledge that this research could not have been possible
[00:05:53.310]without a support of the grants from the National Science Foundation,
[00:05:57.590]the Nebraska Center for Energy Sciences Research and the UNL UCARE
[00:06:01.940]program. And I would like to thank Dr. Niu
[00:06:05.420]for mentoring and guiding me through this project.
[00:06:11.030]Thank you all for listening and have a great rest of your day.

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Structure-Guided Engineering of A Short-Chain Alcohol Dehydrogenase for Asymmetric Synthesis

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