An unusual metal-bound 4-fluorothreonine transaldolase from Streptomyces sp. MA37 catalyses promiscuous transaldol reactions

Abstract

β-Hydroxy-α-amino acids (βH-AAs) are key components of many bioactive molecules as well as exist as specialised metabolites. Among these βH-AAs, 4-fluorothreonine (4-FT) is the only naturally occurring fluorinated AA discovered thus far. Here we report overexpression and biochemical characterisation of 4-fluorothreonine transaldolase from Streptomyces sp. MA37 (FTaseMA), a homologue of FTase previously identified in the biosynthesis of 4-FT in S. cattleya. FTaseMA displays considerable substrate plasticity to generate 4-FT as well as other β-hydroxy-α-amino acids with various functionalities at C4 position, giving the prospect of new chemo-enzymatic applications. The enzyme has a hybrid of two catalytic domains, serine hydroxymethyltransferase (S) and aldolase (A). Site-directed mutagenesis allowed the identification of the key residues of FTases, suggesting that the active site of A domain has a historical reminiscent feature in metal-dependent aldolases. Elemental analysis demonstrated that FTaseMA is indeed a Zn 2+ -dependent enzyme, the first example of pyridoxal phosphate (PLP) enzyme family fused with a metal-binding domain carrying out a distinct catalytic role. Finally, FTaseMA showed divergent evolutionary origin with other PLP dependent enzymes.

Discovery of New Antibacterial Accramycins from a Genetic Variant of the Soil Bacterium, Streptomyces sp. MA37

“…During our natural product screening program, we found that the soil isolate, Streptomyces sp. MA37, is a highly prolific producer of SMs with great chemical diversity, for example, pyrrolizidines [ 11 ], carbazoles [ 12 , 13 , 14 , 15 , 16 ], siderophores [ 17 , 18 ], and fluorinated compounds [ 19 , 20 , 21 , 22 , 23 , 24 ]. More recently, we discovered a new fasamycin congener, accramycin A 1 , as one of the minor SMs in the MA37 wild type (WT) strain (0.2 mg/L) [ 25 ].…”

Defluorination of 4-fluorothreonine by threonine deaminase

“…MA37 and characterized its function in the last step of the transaldol reaction of 4-FT biosynthesis. It utilizes L-Thr and fluoroacetaldehyde (FAd) as substrates and pyridoxal phosphate (PLP) as a cofactor to generate 4-FT. 24 To test whether LAAO acts on 4-FT 2, we performed a coupling reaction. An assay of LAAO with FTaseMA using L-Thr and fluoroacetaldehyde (FAd) to generate 4-FT 2 in situ was carried out (Fig.…”

“…To examine what products were generated during the defluorination catalysed by TDA, we applied the derivatization analytical method that we used in our previous study. 24 2,4-Dinitrophenylhydrazine (DNPH) was used as a derivatization agent for monitoring aldehyde and ketone species in the reaction mixture. The reaction mixture was derivatized by DNPH, followed by HPLC analysis of keto-DNPH derivatives.…”

Identification of 5-Fluoro-5-Deoxy-Ribulose as a Shunt Fluorometabolite in Streptomyces sp. MA37

“…In particular, MA37 produces 1 , 2 and a series of unidentified fluorinated metabolites as observed in 19 F-NMR analysis of the supernatant of the culture broth of MA37 [ 11 ]. It has been shown that 1 and 2 in MA37 originate from the same biosynthetic pathway as the one in S. cattleya [ 11 , 16 ]. A combination of chemical synthesis, bioinformatic analysis and biochemical assays allowed the discovery of 3 as a new fluorometabolite and the identification of the second fdr biosynthetic gene cluster (BGC) in MA37 [ 2 ].…”