MenD from Bacillus subtilis: A Potent Catalyst for the Enantiocomplementary Asymmetric Synthesis of Functionalized α‐Hydroxy Ketones

Abstract

The thiamine diphosphate‐dependent enzyme 2‐succinyl‐5‐enolpyruvyl‐6‐hydroxy‐3‐cyclohexene‐1‐carboxylate synthase (MenD) catalyzes a Stetter‐like 1,4‐addition of α‐ketoglutarate to isochorismate in the biosynthesis of menaquinone (vitamin K). Here, we describe the carboligation potential of MenD from Bacillus subtilis (BsMenD) for the nonphysiological 1,2‐addition of decarboxylated α‐ketoglutarate (succinylsemialdehyde) and various benzaldehyde derivatives. Furthermore, we engineer BsMenD variants for the enantiocomplementary asymmetric synthesis of functionalized α‐hydroxy ketones. Wild type BsMenD shows an excellent chemo‐ as well as high (R)‐selectivity for the carboligation of α‐ketoglutarate as the donor, and different benzaldehyde derivatives as acceptor yielding (R)‐α‐hydroxy ketones with up to >99 % ee. By engineering (S)‐selective BsMenD variants, based on the recently developed S‐pocket concept, we provide access to most of the corresponding (S)‐α‐hydroxy ketones with up to 98 % ee. In particular, benzaldehyde and meta‐substituted derivatives were converted with high enantioselectivities (ee of 91–98 % (S)). The significantly higher (S)‐selectivity of BsMenD variants than recently published MenD variants from Escherichia coli, could be attributed to a second‐shell residue next to the S‐pocket. A glycine residue, adjacent to the major S‐pocket residues I476 and F477 (standard numbering), is assumed to result in higher structural flexibility in the S‐pocket region of BsMenD, which in turn could result in improved stabilization of the antiparallel orientation of the acceptor.

A Tailor‐Made Chimeric Thiamine Diphosphate Dependent Enzyme for the Direct Asymmetric Synthesis of (S)‐Benzoins

“…These results confirm our findings from recent studies on 2‐succinyl‐5‐enolpyruvyl‐6‐hydroxy‐3‐cyclohexene‐1‐carboxylate synthase (MenD), which revealed that meta ‐substituted benzaldehydes are converted with higher S ‐selectivity and conversion than ortho‐ and para ‐substituted benzaldehydes and unsubstituted benzaldehyde 8. 9 However, the reasons for the preference for meta ‐substituted benzaldehydes in S ‐selective carboligation reactions and the switch in stereoselectivity in the case of ortho ‐ and para ‐substituted benzaldehydes are still not understood. In this case, a combination of steric and electronic interactions between the substrate and the S ‐pocket might influence the stereoselectivity.…”

New Stetter reactions catalyzed by thiamine diphosphate dependent MenD from E. coli

“…In addition to testing further enzymes, protein engineering of ThDP-dependent enzymes might enable a broadening of the donor and the acceptor substrate ranges of Stetter reactions. This has already been quite successfully employed in the stereoselective control of 1,2-additions (Hailes et al, 2013;Westphal et al, 2013aWestphal et al, , 2013bWestphal et al, , 2014. In summary, several new Stetter reactions catalyzed by MenD have been identified which might prove useful in the diversity-oriented production of important building blocks.…”

“…2.2.1.9) catalyzes as a physiological reaction a Stetter-like addition of (decarboxylated) ␣-ketoglutarate to isochorismate (1) ultimately resulting in the formation of SHCHC (2) (Jiang et al, 2007;Dawson et al, 2008Dawson et al, , 2010) (Scheme 1C). MenD from Escherichia coli K12 and Bacillus subtilis has been described and analyzed in detail for its carboligation activity, especially with respect to asymmetric 1,2-additions (Kurutsch et al, 2009;Westphal et al, 2013aWestphal et al, , 2013bWestphal et al, , 2014. MenD is highly specific for its physiological donor substrate ␣-ketoglutarate, but has a broad substrate range with respect to the acceptor aldehydes in 1,2-additions.…”

Towards a Mechanistic Understanding of Factors Controlling the Stereoselectivity of Transketolase

“…For the structural family of ThDP‐dependent DCs this simple two‐state model describes the situation sufficiently and was shown to be highly predictive for the rational design of S‐selective enzyme variants. This could be successfully demonstrated by inverting the stereoselectivity of five different ThDP‐dependent DCs . A standard numbering scheme for DCs facilitated identification and annotation of functionally relevant residues, including the comparison of stereoselectivity‐determining positions …”

“…This could be successfully demonstratedb yi nverting the stereoselectivity of five different ThDP-dependentDCs. [10][11][12][13][14] Astandard numberingscheme for DCs facilitated identification and annotation of functionally relevant residues,i ncluding the comparison of stereoselectivity-determiningp ositions. [15] The relation of the DC-family with the furthere ight structural subfamilies of ThDP-dependente nzymes [4] is characterised by generally low sequence identity [16] but two conserved struc-As tructuralm odel for thiamine-diphosphate (ThDP)-dependent transketolase (TK) was developed to analyse the effect of amino acid exchanges on the stereoselectivity of this synthetically important class of enzymes.I nt his study the carboligation of 3-hydroxypyruvatea sadonora nd propanal, as well as pentanal, wass tudied.…”