ABSTRACT
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 mirabilisPmi LAAD. 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.
KEYWORDS: L-amino acid deaminase; enzyme engineering; α-amino acids; α-keto acids; proton transfer distance INTRODUCTION
α-Keto acids contain both a carboxyl and a keto bifunctional group(Cooper et al. 1983; .Li et al. 2019). They are widely used in pharmaceutical(Wang et al. 2019), feed(Manjarinet al. 2020), food(Song et al. 2016), and chemical(Cooperet al. 1983; Ogo et al. 2004; Xie et al. 2019) synthesis. For instance, α-keto acid tablets can be used as therapeutic agents against nephropathy (e.g., uremia and nitrogen accumulation disorders)(Wang et al. 2019), as feed additive (e.g., α-ketoisocaproic or α-ketoisovaleric acid) to stimulate the growth of animal muscle(Manjarin et al. 2020), as precursor in indole-3-acetic acid synthesis(Song et al. 2016), and for the production of chemicals (e.g., heterocyclic compounds(Ogo et al.2004), n-butanol(Ayodele et al. 2020), and D-amino acids(Liuet al. 2020)). Consequently, chemical(Cooper et al. 1983; Jin et al. 2021; Zhao et al. 2020), fermentative(Vogtet al. 2015), and biocatalytic methods(Jambunathan et al.2014; Liu et al. 2013; Pei et al. 2020) have been developed to ensure a sufficient supply of α-keto acids to meet market demand. Biocatalytic methods have attracted particular attention owing to their short reaction period, high conversion rates, and environmental-friendly processes.
Currently, enzymatic synthesis of α-keto acids relies on the use of amino acid transaminase (AAT, EC 2.1.1.X)(Taylor et al. 1998; Wuet al. 2018), L-amino acid dehydrogenase (LAADH, EC 1.4.1.5)(Jambunathan et al. 2014; Wakamatsu et al. 2017), amino acid oxidase (DAAO, EC 1.1.3.3; LAAO, EC 1.4.3.3)(Asano et al. 2019; Hossain et al. 2014a; Mattevi et al. 1996; Nakano et al. 2019) , and L-amino acid deaminase (LAAD, EC 1.4.3.2)(Ju et al. 2017; Ju et al. 2016; Liu et al.2013; Molla et al. 2017; Motta et al. 2016; Pei et al. 2020; Song et al. 2016; Wang et al. 2019; Wu et al. 2020) (Schemes 1 and 2). However, all of these enzymes suffer from considerable limitations: AAT and LAADH present low conversion rates due to simultaneous reversible reactions; AAT-catalyzed transamination requires additional amino acceptors (e.g., α-ketoglutaric acid)(Tayloret al. 1998); LAADH requires NAD, a costly compound, as a cofactor(Wakamatsu et al. 2017), as well as complex separation and purification of the products; and LAAO-catalyzed deamination releases toxic byproducts (e.g., H2O2) and the difficulties in its recombinant production(Hossain et al.2014a); DAAT and DAAO can also produce keto acids, but the substrates involved are expensive unnatural amino acids. Therefore, these enzymes are not suitable for the industrial production of α-keto acids. In contrast, LAAD-catalyzed deamination reactions do not require any additional cofactors or amino receptors, and do not release toxic by-products. The crystal structure (Pma LAAD (Motta et al.2016) and Pv LAAD (Ju et al. 2016)) first reported laid the foundation for later mechanistic elucidation and protein engineering. It was found that the structure of LAAD was composed of three parts: substrate binding domain (SBD), FAD binding domain gate (FBD) and insertion module(Molla et al. 2017). On this basis, various LAAD variants have been engineered. Li et al. developed a one-step process for KIV production via expressing P. myxofaciens LAAD in E. coli BL21(DE3) and enhanced the yield of KIV (8.2 g/L) by site-directed saturation mutagenesis of LAAD to obtain mutants F318T and N100H (Liet al. 2017). 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 LAADT436/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(Yuan et al. 2019). ThePmi LAADF93S/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). As can be seen, there is room for further improving LAAD catalytic efficiency and in particular, for gearing it toward more than one substrate. Overall, protein engineering should focus on developing more efficient and general-purpose LAAD variants.
In the present study, an efficient and universal whole-cell catalyst for α-keto acids production, with excellent catalytic efficiency and wide substrate scope, was constructed through rational protein engineering and a multi-enzyme cascade strategy. First, the catalytic mechanism of LAAD was analyzed and the key factors affecting catalytic efficiency were identified. Then, a protein engineering method, coupled with screening, allowed for the selection of two variants with high catalytic efficiency toward different amino acids. Finally, a one-pot two-enzyme cascade strategy was applied by co-expressing the two variant LAADs inEscherichia coli .