Title: Attempts to develop an enzyme converting DHIV to KIV

Abstract

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 withmore » the desired activity were obtained.« less

Authors:

Oki, Kenji ^[1]; Lee, Frederick S. ^[2]; Mayo, Stephen L. ^[3]; Daggett, ed., Valerie

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+ Show Author Affiliations

1. Division of Biology and Biological Engineering, California Institute of Technology, 1200 E. California Blvd., MC 114-96, Pasadena, CA 91125, USA, Science & Innovation Center, Mitsubishi Chemical Corporation, Yokohama 227-8502, Japan
2. Protabit LLC, 1010 Union St., Suite 110, Pasadena, CA 91101, USA
3. Division of Biology and Biological Engineering, California Institute of Technology, 1200 E. California Blvd., MC 114-96, Pasadena, CA 91125, USA, Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA

Publication Date:

2019-12-23

Sponsoring Org.:

USDOE

OSTI Identifier:

1580283

Grant/Contract Number:  

SC0011396

Resource Type:

Published Article

Journal Name:

Protein Engineering, Design and Selection

Additional Journal Information:

Journal Name: Protein Engineering, Design and Selection; Journal ID: ISSN 1741-0126

Publisher:

Oxford University Press

Country of Publication:

United Kingdom

Language:

English