Production of α‐Ketoisocaproate and α‐Keto‐β‐Methylvalerate by Engineered L‐Amino Acid Deaminase

Yuan, Yuxiang; Song, Wei; Liu, Jia; Chen, Xiulai; Luo, Qiuling; Li, Liming

doi:10.1002/cctc.201900259

Cited by 16 publications

(15 citation statements)

References 39 publications

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“…This approach is in line with a recent trend in protein engineering that focuses on construction of small and smart libraries while reducing the size of the mutation library and improving evolution efficiency (Li, Qu, Sun, & Reetz, 2019;Sun, Lonsdale, Ilie, Li, & Reetz, 2016). Compared to traditional mutagenesis, this method is more rational as it relies on tunnel identification (Song et al, 2020;Yuan et al, 2019), conformational dynamics (Yang et al, 2017), specific hotspot scanning (Xu , Cen, Singh, Fan, & Wu, 2019;, and saturation mutagenesis. Overall, this protein engineering strategy could greatly improve the performance of enzymes with a release channel or lid structure.…”

Section: Discussionmentioning

confidence: 64%

Rational design of a highly efficient catalytic system for the production of 3′-phosphoadenosine-5′-phosphosulfate from ATP

Liu¹,

Chen²,

Zhong³

et al. 2021

Preprint

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The compound 3′-phosphoadenosine-5′-phosphosulfate (PAPS) serves as a sulfate group donor in the production of valuable sulfated compounds, such as glycosaminoglycan and oxamniquine. However, elevated costs and low conversion efficiency limit the industrial applicability of PAPS. Here, we designed and constructed an efficient and controllable catalytic system for the conversion of ATP (disodium salt) into PAPS without inhibition from by-products. In vitro and in vivo testing in Escherichia coli identified adenosine-5′-phosphosulfate kinase from Penicillium chrysogenum (PcAPSK) as the rate-limiting enzyme. Based on analysis of the catalytic steps and molecular dynamics simulations, a mechanism-guided “ADP expulsion” strategy was developed to generate an improved PcAPSK variant (L7), with a specific activity of 48.94 U·mg-1 and 73.27-fold higher catalytic efficiency (kcat/Km) that of the wild-type enzyme. The improvement was attained chiefly by reducing the ADP-binding affinity of PcAPSK, as well as by changing the enzyme’s flexibility and lid structure to a more open conformation. By introducing PcAPSK L7 in an in vivo catalytic system, 73.59 mM (37.32 g·L-1) PAPS was produced from 150 mM ATP in 18.5 h using a 3-L bioreactor. The achieved titer is the highest reported to date and corresponds to a 98.13% conversion rate. The proposed strategy will facilitate industrial production of PAPS as well as the engineering of similar enzymes.

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Section: Discussionmentioning

confidence: 64%

Rational design of a highly efficient catalytic system for the production of 3′-phosphoadenosine-5′-phosphosulfate from ATP

Liu¹,

Chen²,

Zhong³

et al. 2021

Preprint

Self Cite

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“…Shortening the catalytic distance D1 improved LAAD efficiency. Previously, the engineering of LAAD had focused on structural alignment, [14e] H-bond analysis, [10c] pocket volume, and steric hindrance, [17] with the intent of minimizing product inhibition or maximizing catalytic efficiency. For example, Pei et al reduced product inhibition caused by α-keto valine (K PI-Val ) from 0.8 to 5.4 mM by changing the H-bond interaction to generated variant PmiLAAD S98A/T105A/S106A/L341A .…”

Section: Discussionmentioning

confidence: 99%

“…[16] The Proteus mirabilis PmiLAAD T436/W438A variant presents an enlarged entrance (1.71 Å) to the access tunnel, which increases its catalytic efficiency toward L-Ile to 98.9 g/L (99.7 % conversion), but this variant only works well for L-Ile. [17] The PmiLAAD F93S/P186A/M394V/F184S variant achieved a 6.6-fold higher specific activity toward L-Phe compared to the wild-type, but substrate loading was limited to 12 mM (2 g/L). [14e] As can be seen, there is room for further improving LAAD catalytic efficiency and in particular, for gearing it toward more than one substrate.…”

Section: Introductionmentioning

confidence: 99%

Enhanced Catalytic Efficiency of L‐amino Acid Deaminase Achieved by a Shorter Hydride Transfer Distance

Zhang²,

Song

et al. 2021

ChemCatChem

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A sustainable enzymatic synthesis of α-keto acids from α-amino acids deamination remains limited by the low catalytic efficiency of L-amino acid deaminase (LAAD, EC 1.4.3.2). In this study, the catalytic distance D1 between the substrate αCÀ H and the cofactor FAD N(5) was identified as the key factor limiting efficiency of Proteus mirabilis PmiLAAD based on elucidation of catalytic mechanism. A protein engineering strategy was developed to shorten the distance D1 and get two variants W1 (exhibited for short-chain aliphatic amino acids and charged amino acids) and W2( exhibited for large aromatic amino acids and sulfur-containing amino acids). The reason is the two variants altered the binding pose of the substrate, αhydrogen was improved to be more perpendicular against the plain of the isoalloxazine ring causing the angle between the substrates' αCÀ H, FAD N(5), and FAD N(10) to approach 90°. Finally, PmiLAAD variants (W1 and W2)were used in one pot in a parallel way, thus obtain strain S3, which exhibited conversion > 90 % and titer > 100 g/L toward six selected substrates. These results provide the basis for improving industrial production of α-keto acids via microbial deamination of α-amino acids.

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“…Shortening the catalytic distance D1 improved LAAD efficiency. Previously, the engineering of LAAD had focused on structural alignment (Wu et al 2020), H-bond analysis (Pei et al2020), pocket volume, and steric hindrance (Yuan et al 2019), with the intent of minimizing product inhibition or maximizing catalytic efficiency. For example, Pei et al reduced product inhibition caused by α-keto valine (K PI -Val ) from 0.8 to 5.4 mM by changing the H-bond interaction to generated variantPmi LAAD S98A/T105A/S106A/L341A (Pei et al2020).…”

Section: Discussionmentioning

confidence: 99%

“…Mutant K104R was screened from P. vulgarisLAAD mutant libraries constructed by epPCR for a 1.3-fold increase in KMTB enzymatic activity, but the yield of KMTB was only 63.6 g/L (Hossainet al 2014b). The Proteus mirabilis Pmi LAAD T436/W438A variant presents an enlarged entrance (1.71Å) to the access tunnel, which increases its catalytic efficiency toward L-Ile to 98.9 g/L (99.7% conversion), but this variant only works well for L-Ile (Yuan et al 2019). ThePmi LAAD F93S/P186A/M394V/F184S variant achieved a 6.6-fold higher specific activity toward L-Phe compared to the wild-type, but substrate loading was limited to 12 mM (2 g/L) (Wuet al 2020).…”

mentioning

confidence: 99%

Enhanced catalytic efficiency and universality of L-amino acid deaminase achieved by a shorter proton transfer distance

Zhang²,

Song

et al. 2021

Preprint

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L-amino acid deaminase (LAAD, EC 1.4.3.2) catalyzes the deamination of α-amino acids. At present, sustainable enzymatic α-keto acids synthesis remains limited by the low catalytic efficiency of wild-type LAADs. In this study, catalytic mechanism was elucidated, and catalytic distance D1 between the substrate αC-H and the cofactor FAD N(5) was identified as the key factor limiting efficiency of Proteus mirabilis PmiLAAD. Shortening the distance via protein engineering improved catalytic efficiency toward six selected amino acids. The two variants with the best catalytic properties were W1, which exhibited a preference for short-chain aliphatic amino acids and charged amino acids, and W2, which showed a preference for large aromatic amino acids and sulfur-containing amino acids. The mutated residues in the two variants altered the binding pose of the substrate, α-hydrogen was improved to be more perpendicular against the plain of the isoalloxazine ring causing the angle between the substrates’ αC-H, FAD N(5), and FAD N(10) to approach 90°, and thus shortened the distance. Finally, W1 and W2 were cascade in one Escherichia coli cell to obtain strain S3, which exhibited conversion >90% and yield >100 g/L toward all selected substrates. These results provide the basis for improving industrial production of α-keto acids via microbial deamination of α-amino acids.

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Production of α‐Ketoisocaproate and α‐Keto‐β‐Methylvalerate by Engineered L‐Amino Acid Deaminase

Cited by 16 publications

References 39 publications

Rational design of a highly efficient catalytic system for the production of 3′-phosphoadenosine-5′-phosphosulfate from ATP

Rational design of a highly efficient catalytic system for the production of 3′-phosphoadenosine-5′-phosphosulfate from ATP

Enhanced Catalytic Efficiency of L‐amino Acid Deaminase Achieved by a Shorter Hydride Transfer Distance

Enhanced catalytic efficiency and universality of L-amino acid deaminase achieved by a shorter proton transfer distance

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