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Attempts to develop an enzyme converting DHIV to KIV

Oki, Kenji and Lee, Frederick S. and Mayo, Stephen L. (2019) Attempts to develop an enzyme converting DHIV to KIV. Protein Engineering, Design and Selection, 32 (6). pp. 261-270. ISSN 1741-0126. https://resolver.caltech.edu/CaltechAUTHORS:20200102-092224643

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Abstract

Dihydroxy-acid dehydratase (DHAD) catalyzes the dehydration of R-2,3-dihydroxyisovalerate (DHIV) to 2-ketoisovalerate (KIV) using an Fe-S cluster as a cofactor, which is sensitive to oxidation and expensive to synthesize. In contrast, sugar acid dehydratases catalyze the same chemical reactions using a magnesium ion. Here, we attempted to substitute the high-cost DHAD with a cost-efficient engineered sugar acid dehydratase using computational protein design (CPD). First, we tried without success to modify the binding pocket of a sugar acid dehydratase to accommodate the smaller, more hydrophobic DHIV. Then, we used a chemically activated substrate analog to react with sugar acid dehydratases or other enolase superfamily enzymes. Mandelate racemase from Pseudomonas putida (PpManR) and the putative sugar acid dehydratase from Salmonella typhimurium (StPutD) showed beta-elimination activity towards chlorolactate (CLD). CPD combined with medium-throughput selection improved the PpManR kcat/KM for CLD by four-fold. However, these enzyme variants did not show dehydration activity towards DHIV. Lastly, assuming phosphorylation could also be a good activation mechanism, we found that mevalonate-3-kinase (M3K) from Picrophilus torridus (PtM3K) exhibited adenosine triphosphate (ATP) hydrolysis activity when mixed with DHIV, indicating phosphorylation activity towards DHIV. Engineering PpManR or StPutD to accept 3-phospho-DHIV as a substrate was performed, but no variants with the desired activity were obtained.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1093/protein/gzz042DOIArticle
ORCID:
AuthorORCID
Mayo, Stephen L.0000-0002-9785-5018
Additional Information:© 2019 The Author(s). Published by Oxford University Press. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model). Received: 23 August 2019; Revision requested: 21 September 2019; Accepted: 01 October 2019; Published: 23 December 2019.
Funders:
Funding AgencyGrant Number
Department of Energy (DOE)DE-SC0011396
Mitsubishi Chemical CorporationUNSPECIFIED
Issue or Number:6
Record Number:CaltechAUTHORS:20200102-092224643
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20200102-092224643
Official Citation:Kenji Oki, Frederick S Lee, Stephen L Mayo, Attempts to develop an enzyme converting DHIV to KIV, Protein Engineering, Design and Selection, Volume 32, Issue 6, June 2019, Pages 261–270, https://doi.org/10.1093/protein/gzz042
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:100445
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:02 Jan 2020 19:40
Last Modified:28 Feb 2020 19:22

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