Structure of the branched-chain keto acid decarboxylase (KdcA) from<i>Lactococcus lactis</i>provides insights into the structural basis for the chemoselective and enantioselective carboligation reaction

Berthold, C.L.; Gocke, Dörte; Wood, M.; Leeper, Finian J.; Pohl, Martina; Schneider, Günter

doi:10.1107/s0907444907050433

Cited by 63 publications

(60 citation statements)

References 29 publications

(47 reference statements)

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“…To the best of our knowledge, no enzymes from thermophilic organismsa re ablet oc atalyze the decarboxylationo fk etoisovalerate with similar activityt o that of KivD or KdcA. [19] Thus, KdcA was selected as the startingt emplate in this study. [17,18] In addition, the crystal structure of KdcA has been solved with good resolution (1.8 )a nd is available from the Protein Data Bank (PDB ID:2 vbf and 2vbg).…”

Section: Resultsmentioning

confidence: 99%

Structure‐Guided Engineering of α‐Keto Acid Decarboxylase for the Production of Higher Alcohols at Elevated Temperature

2018

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Branched-chain keto acid decarboxylases (KDCs) are a class of enzymes that catalyze the decarboxylation of α-keto acids. They are key enzymes for production of higher alcohols in vivo and in vitro. However, the two most active KDCs (KivD and KdcA) have only moderate thermostability (<55 °C), which hinders the production of alcohols at high temperatures. Herein, structure-guided engineering toward improved thermostability of KdcA is outlined. Strategies such as stabilization of the catalytic center, surface engineering, and optimization of dimer interactions were applied. With seven amino acid substitutions, variant 7M.D showed an increase of the temperature at which 50 % of activity remains after one-hour incubation by 14.8 °C without compromising its substrate specificity. 7M.D exhibited greater than 400-fold improvement of half-life at 70 °C and greater than 600-fold increase in process stability in the presence of 4 % isobutanol at 50 °C. 7M.D is more promising for the production of higher alcohols in thermophiles (>65 °C) and in cell-free applications.

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Section: Resultsmentioning

confidence: 99%

Structure‐Guided Engineering of α‐Keto Acid Decarboxylase for the Production of Higher Alcohols at Elevated Temperature

2018

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“…To determine the potential mechanisms by which the mutations identified via directed evolution (Q34H, A290 V and S386P) conferred thermostability, a model of the Kivd protein was acquired via homology modeling 26 by using the crystal structure of L. lactis branched-chain keto acid decarboxylase 27 (PDB ID: 2VBF) which shares an 88% sequence identity with the wild-type Kivd. The acquired Kivd model was visualized and further studied using Pymol software (Figure 4).…”

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confidence: 99%

Engineering a Thermostable Keto Acid Decarboxylase Using Directed Evolution and Computationally Directed Protein Design

Soh¹,

Mak

Lin³

et al. 2017

ACS Synth. Biol.

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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 lignocellulosic thermophiles. KEYWORDS: thermostability, keto acid decarboxylase, high-throughput screening, protein design, directed evolution, isobutyl alcohol K eto acid decarboxylases (Kdc) are an important group of

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“…However, the C2α chiral center appears considerably flattened from an ideal tetrahedron (Figure S2, Table S1 in the Supporting Information). This observation has some precedent in ThDP enzyme crystallography [branched chain keto acid decarboxylase (40), oxalylCoA decarboxylase (41), the E1 component of the pyruvate dehydrogenase complex from Escherichia coli (42), pyruvate oxidase (43), transketolase (44)]. The crystal structure of a small molecule analogue of HBThDP suggests some sp 2 character at the C2α position (45).…”

Section: Discussionmentioning

confidence: 91%

Detection and Time Course of Formation of Major Thiamin Diphosphate-Bound Covalent Intermediates Derived from a Chromophoric Substrate Analogue on Benzoylformate Decarboxylase

et al. 2009

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The mechanism of the enzyme benzoylformate decarboxylase (BFDC), which carries out a typical thiamin diphosphate (ThDP)-dependent nonoxidative decarboxylation reaction, was studied with the chromophoric alternate substrate (E)-2-oxo-4(pyridin-3-yl)-3-butenoic acid (3-PKB). Addition of 3-PKB resulted in the appearance of two transient intermediates formed consecutively, the first one to be formed a predecarboxylation ThDP-bound intermediate with λmax at 477 nm, and the second one corresponding to the first postdecarboxylation intermediate the enamine with λmax at 437 nm. The time course of formation/depletion of the PKB–ThDP covalent complex and of the enamine showed that decarboxylation was slower than formation of the PKB–ThDP covalent adduct. When the product of decarboxylation 3-(pyridin-3-yl)acrylaldehyde (PAA) was added to BFDC, again an absorbance with λmax at 473 nm was formed, corresponding to the tetrahedral adduct of PAA with ThDP. Addition of well-formed crystals of BFDC to a solution of PAA resulted in a high resolution (1.34 Å) structure of the BFDC-bound adduct of ThDP with PAA confirming the tetrahedral nature at the C2α atom, rather than of the enamine, and supporting the assignment of the λmax at 473 nm to the PAA–ThDP adduct. The structure of the PAA–ThDP covalent complex is the first example of a product–ThDP adduct on BFDC. Similar studies with 3-PKB indicated that decarboxylation had taken place. Evidence was also obtained for the slow formation of the enamine intermediate when BFDC was incubated with benzaldehyde, the product of the decarboxylation reaction thus confirming its presence on the reaction pathway.

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Structure of the branched-chain keto acid decarboxylase (KdcA) fromLactococcus lactisprovides insights into the structural basis for the chemoselective and enantioselective carboligation reaction

Cited by 63 publications

References 29 publications

Structure‐Guided Engineering of α‐Keto Acid Decarboxylase for the Production of Higher Alcohols at Elevated Temperature

Structure‐Guided Engineering of α‐Keto Acid Decarboxylase for the Production of Higher Alcohols at Elevated Temperature

Engineering a Thermostable Keto Acid Decarboxylase Using Directed Evolution and Computationally Directed Protein Design

Detection and Time Course of Formation of Major Thiamin Diphosphate-Bound Covalent Intermediates Derived from a Chromophoric Substrate Analogue on Benzoylformate Decarboxylase

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