Acid Racemase Research Articles

Identification and Characterization of D‐Succinylase, and a Proposed Enzymatic Method for D‐Amino Acid Synthesis

AbstractChiral amino acids are important intermediates for the pharmaceutical industry. We have developed a novel one‐pot enzymatic method for D‐amino acid synthesis by the dynamic kinetic resolution of N‐succinyl‐dl‐amino acids using D‐succinylase (DSA) and N‐succinylamino acid racemase (NSAR, EC 4.2.1.113). The DSA from Cupriavidus sp. P4‐10‐C, which hydrolyzes N‐succinyl‐D‐amino acids enantioselectively to their corresponding D‐amino acids, was identified for the first time by screening soil microorganisms. Subsequently, the DSA gene was cloned and overexpressed in Escherichia coli. DSA was shown to comprise two subunits with molecular masses of 26 kDa and 60 kDa. Additionally, the NSAR gene from Geobacillus stearothermphilus NCA1503, which racemizes N‐succinylamino acids, was also cloned and overexpressed in E. coli. The highly purified DSA and NSAR prepared from each recombinant E. coli were characterized and used for D‐amino acid synthesis. A one‐pot enzymatic method converted 100 mM N‐succinyl‐dl‐phenylalanine to D‐phenylalanine in 91.1% conversion with 86.7% ee. This novel enzymatic method may be useful for the industrial production of many D‐amino acids.magnified image

Rational re-design of the “double-racemase hydantoinase process” for optically pure production of natural and non-natural l-amino acids

The “hydantoinase process” is a well-established method for the industrial production of optically pure d-amino acids. However, due to the strict d-enantioselectivity of most hydantoinase enzymes, the process is less efficient for l-amino acid production. We present a new chemo-enzymatic cascade reaction for natural and non-natural l-amino acid production from racemic mixtures of 5-monosubstituted hydantoins. This system comprised the following enzymes: d-hydantoinase from Agrobacterium tumefaciens BQL9, hydantoin racemase 1 from A. tumefaciens C58 and l-N-carbamoylase from Geobacillus stearothermophilus CECT43, together with N-succinyl-amino acid racemase from G. kaustophilus CECT4264. This latter presents catalytic promiscuity and racemizes N-carbamoyl-amino acids. This activity avoids the accumulation of N-carbamoyl-d-amino acid in the reaction due to the strict d-enantioselectivity of the hydantoinase. The optimum pH for the system proved to be 8.0, whereas optimum temperature range was 50–65°C, with the maximum reaction rate at 60°C. The metal ion cobalt was added directly to the reaction mixture (end concentration 1mM), but in the case of d-hydantoinase, overexpression in presence of 0.5mM Co2+ was also necessary. The enzymatic cascade reaction produced different optically pure l-amino acids by dynamic kinetic resolution, achieving 100% conversion even at high substrate concentrations (100mM) with no noticeable inhibition. This total conversion demonstrates that the “double-racemase hydantoinase process” upgrades the classical “hydantoinase process” for natural and non-natural l-amino acid production.

Biochemical and Mutational Characterization of N-Succinyl-Amino Acid Racemase from Geobacillus stearothermophilus CECT49.

N-Succinyl-amino acid racemase (NSAAR), long referred to as N-acyl- or N-acetyl-amino acid racemase, is an enolase superfamily member whose biotechnological potential was discovered decades ago, due to its use in the industrial dynamic kinetic resolution methodology first known as "Acylase Process". In previous works, an extended and enhanced substrate spectrum of the NSAAR from Geobacillus kaustophilus CECT4264 toward different N-substituted amino acids was reported. In this work, we describe the cloning, purification, and characterization of the NSAAR from Geobacillus stearothermophilus CECT49 (GstNSAAR). The enzyme has been extensively characterized, showing a higher preference toward N-formyl-amino acids than to N-acetyl-amino acids, thus confirming that the use of the former substrates is more appropriate for a biotechnological application of the enzyme. The enzyme showed an apparent thermal denaturation midpoint of 77.0 ± 0.1 °C and an apparent molecular mass of 184 ± 5 kDa, suggesting a tetrameric species. Optimal parameters for the enzyme activity were pH 8.0 and 55-65 °C, with Co(2+) as the most effective cofactor. Mutagenesis and binding experiments confirmed K166, D191, E216, D241, and K265 as key residues in the activity of GstNSAAR, but not indispensable for substrate binding.

Enzymatic dynamic kinetic resolution of racemic N-formyl- and N-carbamoyl-amino acids using immobilized L-N-carbamoylase and N-succinyl-amino acid racemase.

Taking advantage of the catalytic promiscuity of L-carbamoylase from Geobacillus stearothermophilus CECT43 (BsLcar) and N-succinyl-amino acid racemase from Geobacillus kaustophilus CECT4264 (GkNSAAR), we have evaluated the production of different optically pure L-α-amino acids starting from different racemic N-formyl- and N-carbamoyl-amino acids using a dynamic kinetic resolution approach. The enzymes were immobilized on two different solid supports, resulting in improved stability of the enzymes in terms of thermostability and storage when compared to the enzymes in solution. The bienzymatic system retained up to 80% conversion efficiency after 20 weeks at 4 °C and up to 90% after 1 week at 45 °C. The immobilization process also resulted in a great enhancement of the activity of BsLcar toward N-formyl-tryptophan, showing for the first time that substrate specificity of L-carbamoylases can be influenced by this approach. The system was effective for the biosynthesis of natural and unnatural L-amino acids (enantiomeric excess (e.e.) >99.5%), such as L-methionine, L-alanine, L-tryptophan, L-homophenylalanine, L-aminobutyric acid, and L-norleucine, with a higher performance toward N-formyl-α-amino acid substrates. Biocatalyst reuse was studied, and after 10 reaction cycles, over 75% activity remained.

Evolution of structure and function in the o ‐succinylbenzoate synthase family

Understanding how protein function and structure evolve is vital for redesigning proteins and utilizing data from genome projects. In pursuit of this goal, we analyzed the evolution of structure and function in the o-succinylbenzoate synthase (OSBS) family, which is in the enolase superfamily. Although OSBSs are extremely divergent and can share < 15% identity, phylogenetic analysis indicates that they have a single evolutionary origin. However, no sequence or structural motifs unique to the OSBS family could be identified; all residues conserved in the OSBS family are also found in enolase superfamily members that have different functions. In addition, the N- and C-terminal domains are oriented differently in three crystallized OSBSs. Thus, although function has been conserved in this family, the precise mode of subtrate binding and determining specificity has not. Included in the OSBS family tree are several uncharacterized proteins that group with the promiscuous OSBS/N-acylamino acid racemase from Amycolatopsis. These probably have additional or alternative functions to OSBS because many are from organisms lacking the pathway in which OSBS is an intermediate. Several of these are promiscuous, like the Amycolatopsis enzyme, supporting the hypothesis that new protein functions evolve through promiscuous intermediates. These are found in taxonomically diverse species, including both Eubacteria and Archaea, suggesting that this new protein function has been transmitted by lateral gene transfer. This work was funded by NIH Grants GM60595 and GM071790.