Streptococcus agalactiae amylomaltase offers insight into the transglycosylation mechanism and the molecular basis of thermostability among amylomaltases

Abstract

Amylomaltase (AM) catalyzes transglycosylation of starch to form linear or cyclic oligosaccharides with potential applications in biotechnology and industry. In the present work, a novel AM from the mesophilic bacterium Streptococcus agalactiae (SaAM), with 18–49% sequence identity to previously reported AMs, was characterized. Cyclization and disproportionation activities were observed with the optimum temperature of 30 °C and 40 °C, respectively. Structural determination of SaAM, the first crystal structure of small AMs from the mesophiles, revealed a glycosyl-enzyme intermediate derived from acarbose and a second acarbose molecule attacking the intermediate. This pre-transglycosylation conformation has never been before observed in AMs. Structural analysis suggests that thermostability in AMs might be mainly caused by an increase in salt bridges since SaAM has a lower number of salt bridges compared with AMs from the thermophiles. Increase in thermostability by mutation was performed. C446 was substituted with A/S/P. C446A showed higher activities and higher kcat/Km values for starch in comparison to the WT enzyme. C446S exhibited a 5 °C increase in optimum temperature and the threefold increase in half-life time at 45 °C, most likely resulting from H-bonding interactions. For all enzymes, the main large-ring cyclodextrin (LR-CD) products were CD24-CD26 with CD22 as the smallest. C446S produced more CD35-CD42, especially at a longer incubation time.

Production of Large-Ring Cyclodextrins by Amylomaltases

“…More common producers of AM are among Thermus species where the optimum temperature is conserved around 60-75 • C [26][27][28][29][30]. It is worth noting that a particular mutation on AM gene resulted in an increase in optimal temperature and half-life, compared to the wild type as illustrated by C446S mutated AM from Streptococcus agalactiae, where the optimum temperature increased by 5 • C from 40 • C and showed 3-fold increase in half-life at 45 • C [31]. Similarly, the mutated A460V enzyme from Corynebacterium glutamicum (CgAM) exhibited 5 • C increase in optimum temperature which made it more thermostable compared to the wild type.…”

“…Figure S1 in Supplementary Materials shows sequence alignment of AMs and D-enzymes. The catalytic triad residues of TaAM consist of D293, E340, and D395 as a nucleophile, acid-base catalyst, and transition state-stabilizer, respectively, as evidenced by covalently bound intermediate of Thermus thermophilus AM (TtAM) [57], Streptococcus agalactiae AM (SaAM) [31], AtDPE1 [53], and potato D-enzyme [54]. The whole substrate-binding tract of AMs was most completely illustrated by co-crystallization of TaAM with 34 mer CA, presenting as an asymmetric dimer [58] (Figure 2A).…”

“…Meanwhile, studies on AMs from mesophilic bacteria [36,63,64] and plants [40] are also increasing. Recently, Tumhom and co-workers reported the biochemical characterization on AM from mesophilic S. agalactiae, where SaAM was observed to catalyze the production of LR-CDs with the DP range of 22 to 50 when using pea starch as substrate [31]. Cyclization reaction to produce LR-CDs involved intramolecular transglycosylation, whereby the reaction conditions, incubation time, substrate, substrate pretreatment, and gene mutagenesis would impact the product yields.…”

Amylomaltases in Extremophilic Microorganisms

“…The mesophilic S. agalactiae amylomaltase was also subjected to mutation experiments [ 25 ]. In particular, C446, which is conserved in other amylomaltases from mesophiles such as E. coli and C. glutamicum , was substituted with a Pro residue, because it is highly conserved in thermophilic amylomaltases (P450 in O87172 in Figure 2 ) and is thought to contribute to enzyme rigidity and stability.…”

Identification of an Amylomaltase from the Halophilic Archaeon Haloquadratum walsbyi by Functional Metagenomics: Structural and Functional Insights

“…For plants, structures have been determined for Arabidopsis thaliana [19] and potato [20]. For bacteria, structures have been obtained for amylomaltases from Aquifex aeolicus (Protein Data Bank, PDB, accession number 1TZ7), Corynebacterium glutamicum [21], E. coli [22], Streptococcus agalactiae [23], Thermus brockianus [24], T. thermophilus AT-62 (formerly indicated as T. aquaticus [25]) [26]. No structural studies are available for amylomaltases from Archaea.…”