New Enzymatic Functions Through Computational Design

Author : Wai Shun Mak
Publisher :
ISBN 13 : 9780355764055
Total Pages : pages
Book Rating : 4.7/5 (64 download)

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Download or read book Computational Design and Mining for Enzyme Functions written by Wai Shun Mak and published by . This book was released on 2017 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Chapter 1: The ability to rationally engineer enzyme function has wide ranging applications and is a rigorous test on our understanding of enzyme function. We used a combination of genomic mining, structural biology, and machine-learning to determine the structural features that enable soluble aldehyde deformylating oxygenase (ADO) activity. These features were then used to introduce ADO activity into the small subunit of ribonucleotide reducatase. The ADO family of enzymes has previously only been observed in a small number of cyanobacteria but its ability to transform linear aldehydes into alkanes has significant implications in bioenergy, synthetic biology and chemoenzymatic synthesis applications. The enzymes discovered here from both genomic mining and engineering efforts yielded enzymes with novel specificity for medium chain aldehydes. In addition, they revealed that this functionality is observed in various species of Bacteria and Archaea and can be readily altered based on the structural features identified. Chapter 2:The ability to biosynthetically produce chemicals beyond what is commonly found in Nature requires the discovery of novel enzyme function. Here we utilize two approaches to discover enzymes that enable specific production of longer-chain (C5–C8) alcohols from sugar. The first approach combines bioinformatics and molecular modeling to mine sequence databases, resulting in a diverse panel of enzymes capable of catalyzing the targeted reaction. The median catalytic efficiency of the computationally selected enzymes is 75-fold greater than a panel of naively selected homologues. This integrative genomic mining approach establishes a unique avenue for enzyme function discovery in the rapidly expanding sequence databases. The second approach uses computational enzyme design to reprogram specificity. Each of these approaches results in enzymes with > 100-fold increase in specificity for the targeted reaction. When enzymes from either approach are integrated in vivo, longer-chain alcohol production increases over 10-fold and represents 95% of the total alcohol products. Chapter 3: Enzyme active sites have been through millions of years of evolution during which their active site microenvironments are fine-tuned. Active site residues are commonly conserved within protein families, indicating their importance for substrate recognition and catalysis. In this work, we systematically mutate active site residues of L-threonine dehydrogenase from Thermoplasma volcanium and characterize the mutants against a panel of substrate analogs. Our results demonstrate that only a subset of these residues play an essential role in substrate recognition and catalysis, and that the native enzyme activity can be further enhanced roughly 6-fold by a single point mutation. Kinetic characterization of mutants on substrate analogs show that L-threonine dehydrogenase possesses promiscuous activities towards other chemically similar compounds previously not observed. Quantum mechanical calculations on these substrates’ hydride donating ability also reveal that this enzyme did not evolve to harness the intrinsic substrate reactivity for enzyme catalysis. Our quantitative analysis provides insights into connections between the details of enzyme active site structure and specific function. These results are directly applicable to rational enzyme design and engineering. Chapter 4: Keto acid decarboxylase (Kdc) is a key enzyme in producing keto acid derived higher alcohols, like isobutanol. The most active Kdc’s are found in mesophiles; the only reported Kdc activity in thermophiles is 2 orders of magnitude less active. Therefore, the thermostability of mesophilic Kdc limits isobutanol production temperature. Here, we report development of a thermostable 2-ketoisovalerate decarboxylase (Kivd) with 10.5-fold increased residual activity after 1h preincubation at 60 °C. Starting with mesophilic Lactococcus lactis Kivd, a library was generated using random mutagenesis and approximately 8,000 independent variants were screened. The top single-mutation variants were recombined. To further improve thermostability, 16 designs built using Rosetta Comparative Modeling were screened and the most active was recombined to form our best variant, LLM4. Compared to wild-type Kivd, a 13 °C increase in melting temperature and over 4-fold increase in half-life at 60 °C were observed. LLM4 will be useful for keto acid derived alcohol production in lignocellulytic thermophiles.