Biocatalytic application of rare PLP-dependent aminotransferases for the synthesis of high value amino acids and amines
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Date
01/07/2019Author
Serpico, Annabel
Metadata
Abstract
Optically active amines and amino acids are ubiquitously distributed in nature where they
play many crucial roles. Moreover, it was recently estimated that around 40% of
blockbuster drugs and 20% of agrochemicals contain chiral amines in their structure, thus
there has been considerable effort in developing efficient, low cost and widely-applicable
methods for their production. Compared to classical chemical synthesis, aminotransferase
(AT) enzymes have been widely explored as a more efficient and sustainable method for
the preparation of optically pure amines from the corresponding ketones. In several cases
these biocatalysts have replaced the existing chemical catalysts, which are limited by their
high cost and poor regio- and stereoselectivity.
Two very interesting, distantly-related class III bacterial ATs have been investigated
in this study: the D-phenylglycine (D-Phg) aminotransferase (D-PhgAT) and the amino-pentol
AT (FumI). The D-PhgAT from Pseudomonas stutzeri ST-201 catalyses the reversible
transamination from L-glutamic acid to benzoylformate, yielding α-ketoglutarate and D-Phg
with high (>99%) enantiomeric excess (% ee). The D-PhgAT possesses the unique feature
that the amino acid donor and amino acid product display an inverted absolute
configuration. Thus, D-PhgAT is a very promising biocatalyst as it yields high value D-amino
acids from inexpensive L-amino acid donors.
By carrying out a detailed kinetic analysis of recombinant D-PhgAT the best
substrates were identified and an optimized method, delivering a range of enantiopure
aromatic D-amino acids at 1 g scale, has been developed. Moreover, the x-ray crystal
structure of D-PhgAT at 2.25 Å resolution has been determined with its pyridoxal 5’-
phosphate (PLP) cofactor bound as an internal aldimine. The active site architecture
highlights various residues potentially involved in catalysis and illuminates the basis of the
exquisite enantioselectivity of this unique member of the AT superfamily. These studies
promote D-PhgAT as a useful tool for the sustainable production of high value, aromatic D-amino
acids.
Fatty amines (C8-C20) are high value products with a wide variety of applications in
fabric softeners, detergents and cosmetic formulations. The unusual Sphingopyxis FumI
detoxifies the C20 mycotoxin natural product fumonisin B1 (FB1). FumI catalyses
transamination at the C2 position of a hydrolyzed fumonisin B1 (HFB1) derivative with
pyruvate as the amino acceptor. A screen of prochiral C2 ketones of varying chain length
(C3-C17 2K) with L-alanine as amino donor has revealed that the recombinant FumI is
capable of producing alkyl-amines of various chain lengths (C3-C17) by working in the
reverse direction. Chiral HPLC analysis has revealed that the enzyme generates (S)-amines
with high ee (>99%). The regioselectivity of FumI was also determined using prochiral keto-substrates
with substitutions along the chain (such as 3-decanone (C10-3K) and 5-decanone
(C10-5K)). Interestingly, C10-3K, but not C10-5K, was converted to the corresponding
amine. A range of long chain aldehydes (C10-C20) were also converted by FumI to the
corresponding amines with L-Ala as the amino donor. The x-ray crystal structure of the PLP-bound,
internal aldimine form of FumI was solved to a resolution of 1.6 Å by molecular
replacement using a Bacillus subtilis AT as a model. The structure suggested residues
potentially involved in catalysis and revealed a potential hydrophobic binding site for the
long alkyl chain substrates. The combination of aldehyde and ketone alkyl chain substrate
promiscuity, the high enantioselectivity and the 3D structures of this rare AT suggests that
FumI is a very useful addition to the biocatalytic toolbox for the synthesis of high value fatty
amines.