Identification of an α-Oxoamine Synthase and a One-Pot Two-Step Enzymatic Synthesis of α-Amino Ketones

Abstract

Alb29, an α-oxoamine synthase involved in albogrisin biosynthesis in Streptomyces albogriseolus MGR072, was characterized and responsible for the incorporation of L-glutamate to acyl-coenzyme A substrates. Combined with Alb29 and Mgr36 (an acyl-coenzyme A ligase), a one-pot enzymatic system was established to synthesize seven α-amino ketones. When these α-amino ketones were fed into the alb29 knockout strain Δalb29, respectively, the albogrisin analogs with different side chains were observed.

Understanding and Circumventing the Requirement for Native Thioester Substrates for α-Oxoamine Synthase Reactions

“…Others have also developed creative solutions to diminish the requirement for stoichiometric CoA. For example, Zhou and co-workers established a one-pot, two-step enzymatic system that allowed for the recycling of the coenzyme A activator required for the synthesis of α-amino ketones . Although the coenzyme A activator is still required, this system avoids the need for stoichiometric acyl-CoA substrate.…”

“…Ideally, enzymes requiring thioester substrates would accept electrophiles with shorter, cheaper activating groups such as N -acetylcysteamine (SNAC, 3 , Figure A) . Many enzymes from polyketide pathways do in fact accept acyl-SNAC thioesters as surrogates for their native ACP substrates. , However, in other cases, employing acyl-SNAC reagents results in little or no product formation, including in reactions involving α-oxamine synthases. , This enzyme class relies on initial condensation of an α-amino acid onto the pyridoxal phosphate (PLP) cofactor to generate an external aldimine intermediate. − This state lowers the p K a of the α-proton significantly, allowing for deprotonation by a proximal basic residue to form a nucleophilic quinonoid intermediate ( 5 , Figure B). The quinonoid can subsequently attack an acyl electrophile, such as acyl-CoA or acyl-ACP substrates.…”

“…An example of the limitation this creates is highlighted in the transformation of Larginine (7) to ketone 8 with propionyl-CoA where a five-gram reaction would cost over $50,000 for the propionyl-CoA alone (Figure 1B). 3 To avoid the requirement for stoichiometric CoA-activated or carrier protein-bound substrates, many groups have employed protein engineering strategies, 4 recycling systems, 5 or the use of simplified, more economical thioester substrates (Figure 1A and Supplementary Figure 4). 6 For example, LovD, an acyltransferase that is a part of a polyketide pathway from Aspergillus terreus, natively uses an ACP domain-bound electrophile for the transformation of monacolin J acid to simvastatin.…”

“…8,9 However, in other cases, employing acyl-SNAC reagents results in little or no product formation, 10 including in reactions involving αoxamine synthases. 5,11 This enzyme class relies on initial condensation of an α-amino acid onto the pyridoxal phosphate (PLP) cofactor to generate an external aldimine intermediate. 12−15 This state lowers the pK a of the α-proton significantly, allowing for deprotonation by a proximal basic residue to form a nucleophilic quinonoid intermediate (5, Figure 1B).…”

Versatile Chemo-Biocatalytic Cascade Driven by a Thermophilic and Irreversible C–C Bond-Forming α-Oxoamine Synthase

“…This substrate scope sets Th AOS apart from other wild-type AOS enzymes, which are typically highly substrate specific, especially for the AA susbtrate . Two recently published AOS biocatalysts, SxtA and Alb29, catalyzed 9 and 7 reactions, respectively, but only with one AA each ( l -Arg for SxtA and l -Glu for Alb29). , A more recent AOS biocatalyst, the fusion enzyme BioWF from Corynebacterium amycolatum , was reported to catalyze 12 unique reactions, mostly with l -Ala but also including two reactions with Gly and l -Ser . Therefore, it appears that Th AOS is unique in terms of the diversity of substrate acceptance for both AA and acyl-CoA thioester substrates.…”