8-Amino-7-oxononanoate synthase (also known as 7-keto-8-aminopelargonate synthase, EC 2.3.1.47) is a pyridoxal 5'-phosphate-dependent enzyme which catalyzes the decarboxylative condensation of L-alanine with pimeloyl-CoA in a stereospecific manner to form 8(S)-amino-7-oxononanoate. This is the first committed step in biotin biosynthesis. The mechanism of Escherichia coli AONS has been investigated by spectroscopic, kinetic, and crystallographic techniques. The X-ray structure of the holoenzyme has been refined at a resolution of 1.7 A (R = 18.6%, R(free) = 21. 2%) and shows that the plane of the imine bond of the internal aldimine deviates from the pyridine plane. The structure of the enzyme-product external aldimine complex has been refined at a resolution of 2.0 A (R = 21.2%, R(free) = 27.8%) and shows a rotation of the pyridine ring with respect to that in the internal aldimine, together with a significant conformational change of the C-terminal domain and subtle rearrangement of the active site hydrogen bonding. The first step in the reaction, L-alanine external aldimine formation, is rapid (k(1) = 2 x 10(4) M(-)(1) s(-)(1)). Formation of an external aldimine with D-alanine, which is not a substrate, is significantly slower (k(1) = 125 M(-)(1) s(-)(1)). Binding of D-alanine to AONS is enhanced approximately 2-fold in the presence of pimeloyl-CoA. Significant substrate quinonoid formation only occurs upon addition of pimeloyl-CoA to the preformed L-alanine external aldimine complex and is preceded by a distinct lag phase ( approximately 30 ms) which suggests that binding of the pimeloyl-CoA causes a conformational transition of the enzyme external aldimine complex. This transition, which is inferred by modeling to require a rotation around the Calpha-N bond of the external aldimine complex, promotes abstraction of the Calpha proton by Lys236. These results have been combined to form a detailed mechanistic pathway for AONS catalysis which may be applied to the other members of the alpha-oxoamine synthase subfamily.

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Enantiomerically pure chiral amines are valuable synthetic intermediates, particularly for the preparation of pharmaceutical compounds. Traditionally, chiral amines have been obtained by resolution-based procedures, for example, by kinetic resolution of a racemate using an enzyme [1,2] or crystallization of a diastereomer using a chiral acid to form a salt. [3] Increasingly, there is a desire to develop asymmetric approaches, or their equivalents, which can in principal deliver the product in 100 % yield and 100 % ee. For example, transaminases have been utilized for the conversion of COMMUNICATIONS

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Enantiomerically pure chiral amines are of increasing value in organic synthesis, especially as resolving agents, [1] chiral auxiliaries/chiral bases, [2] and catalysts for asymmetric synthesis. [3] In addition, chiral amines often possess pronounced biological activity in their own right and hence are in demand as intermediates for agrochemicals and pharmaceuticals. [4] Current methods for the preparation of enantiomerically pure chiral amines are largely based upon the resolution of racemates, either by recrystallization of diastereomeric salts [5] or by enzyme-catalyzed kinetic resolution of racemic substrates using lipases and acylases.[6] To develop more efficient methods, attention is turning towards asymmetric approaches or their equivalent, for example, the asymmetric hydrogenation of imines [7] or the conversion of ketones into amines by using transaminases.[8] Attempts to develop dynamic kinetic resolutions, which employ enzymes in combination with transition-metal catalysts, have unfortunately been hampered by the harsh conditions required to racemize amines.[9]Recently we reported a novel catalytic method for the preparation of optically active chiral amines by deracemization of the corresponding racemic mixture (Figure 1).[10] The deracemization approach relies upon coupling an enantioselective amine oxidase with a nonselective reducing agent to effect stereoinversion of the S to R enantiomer via the intermediate achiral imine.The S enantiomer selective amine oxidase used for the deracemization of (R/S)-a-methylbenzyl amine was identified from a library of variants of the wild-type enzyme, from Aspergillus niger, by using a high-throughput colorimetric screen to guide selection.[10] The library of variants was generated by randomly mutating the plasmid harboring the amine oxidase gene by using the E. coli XL1-Red mutator strain. Using (S)-a-methylbenzylamine as the target substrate we were able to identify a variant (Asn336Ser) that possessed significantly improved catalytic activity (47 fold) and enantioselectivity (sixfold) towards this particular substrate compared to the wild type enzyme. To explore the opportunities for using this variant amine oxidase to deracemize other racemic chiral amines we decided to undertake a more detailed study of its substrate specificity. Herein we show that the Asn336Ser variant possesses broad substrate specificity and high enantioselectivity towards a wide range of chiral amines.Prior to carrying out further studies with the Asn336Ser amine oxidase, an additional mutation was introduced into the sequence (Met348Lys) that resulted in a variant enzyme (hereafter referred to as Asn336Ser) with higher specific activity and expression levels although its substrate specificity appeared unchanged (data not shown). Incorporation of an N-terminal histidine tag into the amine oxidase allowed facile purification of both the wild-type and Asn336Ser variant in one step, by a nickel-affinity column, to yield protein of > 90 % purity as evidenced by gel electrophoresis (Figure ...