Molecular engineering of industrial enzymes: recent advances and future prospects

Yang, Haiquan; Li, Jianghua; Shin, Hyun‐Dong; Du, Guocheng; Liu, Long; Chen, Jian

doi:10.1007/s00253-013-5370-3

Cited by 65 publications

(42 citation statements)

References 59 publications

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“…We however considered it unlikely that they would show generally improved chaperone activity for at least two reasons. First, in a wide variety of laboratory evolution experiments where variant enzymes are selected that show improved activity against one substrate, often though not always show decreased activity against other unrelated substrates (Goldsmith et al, 2012; Yang et al, 2013). More specifically, other efforts at improving chaperone activity, though showing some success in generating mutants that were better with the substrates they were selected on, in general showed decreased chaperone activity against other substrates (Wang et al, 2002; Aponte et al, 2010; Schweizer et al, 2011).…”

Section: Resultsmentioning

confidence: 99%

Super Spy variants implicate flexibility in chaperone action

Qin

Wang

Petrotchenko

et al. 2014

eLife

104

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Experimental study of the role of disorder in protein function is challenging. It has been proposed that proteins utilize disordered regions in the adaptive recognition of their various binding partners. However apart from a few exceptions, defining the importance of disorder in promiscuous binding interactions has proven to be difficult. In this paper, we have utilized a genetic selection that links protein stability to antibiotic resistance to isolate variants of the newly discovered chaperone Spy that show an up to 7 fold improved chaperone activity against a variety of substrates. These “Super Spy” variants show tighter binding to client proteins and are generally more unstable than is wild type Spy and show increases in apparent flexibility. We establish a good relationship between the degree of their instability and the improvement they show in their chaperone activity. Our results provide evidence for the importance of disorder and flexibility in chaperone function.DOI: http://dx.doi.org/10.7554/eLife.01584.001

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Section: Resultsmentioning

confidence: 99%

Super Spy variants implicate flexibility in chaperone action

Qin

Wang

Petrotchenko

et al. 2014

eLife

104

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“…Ultimately, structural information on the various aromatic acid decarboxylases should prove useful in the engineering of modified versions of these enzymes (42)(43)(44). Initial studies of bacterial phenolic acid decarboxylases with different substrate specificities showed that the generation of chimeric enzymes could be used to alter product profiles (44).…”

Section: Discussionmentioning

confidence: 99%

Structure and Mechanism of Ferulic Acid Decarboxylase (FDC1) from Saccharomyces cerevisiae

Bhuiya

Lee

Jez

et al. 2015

Appl Environ Microbiol

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The nonoxidative decarboxylation of aromatic acids occurs in a range of microbes and is of interest for bioprocessing and metabolic engineering. Although phenolic acid decarboxylases provide useful tools for bioindustrial applications, the molecular bases for how these enzymes function are only beginning to be examined. Here we present the 2.35-Å-resolution X-ray crystal structure of the ferulic acid decarboxylase (FDC1; UbiD) from Saccharomyces cerevisiae. FDC1 shares structural similarity with the UbiD family of enzymes that are involved in ubiquinone biosynthesis. The position of 4-vinylphenol, the product of p-coumaric acid decarboxylation, in the structure identifies a large hydrophobic cavity as the active site. Differences in the ␤2e-␣5 loop of chains in the crystal structure suggest that the conformational flexibility of this loop allows access to the active site. The structure also implicates Glu285 as the general base in the nonoxidative decarboxylation reaction catalyzed by FDC1. Biochemical analysis showed a loss of enzymatic activity in the E285A mutant. Modeling of 3-methoxy-4-hydroxy-5-decaprenylbenzoate, a partial structure of the physiological UbiD substrate, in the binding site suggests that an ϳ30-Å-long pocket adjacent to the catalytic site may accommodate the isoprenoid tail of the substrate needed for ubiquinone biosynthesis in yeast. The three-dimensional structure of yeast FDC1 provides a template for guiding protein engineering studies aimed at optimizing the efficiency of aromatic acid decarboxylation reactions in bioindustrial applications.T he chemical production of benzenoids, such as styrene, from petroleum provides a wide range of building blocks for use in paints, dyes, plastics, and synthetic pharmaceuticals. Current styrene production involves the energy-intensive dehydrogenation of petroleum-derived ethylbenzene and yields more than 30 million tons of material each year (1). As an alternative to petroleumbased synthesis, research into finding renewable sources of benzenoid compounds in nature has focused attention on multiple plant and microbial pathways. Substituted cinnamic acids are abundant molecules in plant lignin polymers and can provide feedstocks for microbial bioprocessing methods aimed at yielding value-added products (2, 3). Degradation of lignin releases ferulic and p-coumaric acids that can be converted to 4-vinylguaicol (4-ethenyl-2-methoxyphenol) and 4-vinylphenol (4-ethenylphenol), respectively, and other hydroxycinnamates can be metabolized to vanillin as a natural flavoring in foods, beverages, and other products (4, 5). Related aromatic compounds are also natural components in wine and other fermented beverages and food (6-8). Similarly, other production routes for catechol and styrene, using microbes that metabolize benzenoid molecules by nonoxidative decarboxylation, have also been explored recently (8-10). For example, overexpression of phenylalanine ammonia lyase from Arabidopsis thaliana and of ferulic acid decarboxylase (FDC) from Saccharomyces cerevis...

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“…They have applications in different industries, namely detergent, food, leather, pharmaceutical, silk, and for recovery of silver from used X-ray films [2,3]. Thermoalkaline proteases represent one of the most commonly used groups of alkaline proteases, particularly because they can function at a pH range of 7-12 and a temperature range of 35-8 complete PDB liquid medium at pH 5.6 containing (g/l): lentil flour, 10; yeast extract, 2; glucose,10; KCl,0.5; KH2PO4, 1; and K2HPO4.…”

Section: Introductionmentioning

confidence: 99%