“…2). Although Escherichia coli FbaA did not epimerize FBP to TBP due to having a small cavity volume, it can epimerize the smaller substrate F6P to T6P 19 .Figure 2Bio-LC diagram and 31 P-NMR spectra for the reaction of F6P with FbaA. ( A ) Bio-LC diagrma of T6P biocoversion from F6P by FbaA.…”
Sugar 4-epimerization reactions are important for the production of rare sugars and their derivatives, which have various potential industrial applications. For example, the production of tagatose, a functional sweetener, from fructose by sugar 4-epimerization is currently constrained because a fructose 4-epimerase does not exist in nature. We found that class II d-fructose-1,6-bisphosphate aldolase (FbaA) catalyzed the 4-epimerization of d-fructose-6-phosphate (F6P) to d-tagatose-6-phosphate (T6P) based on the prediction via structural comparisons with epimerase and molecular docking and the identification of the condensed products of C3 sugars. In vivo, the 4-epimerization activity of FbaA is normally repressed. This can be explained by our results showing the catalytic efficiency of d-fructose-6-phosphate kinase for F6P phosphorylation was significantly higher than that of FbaA for F6P epimerization. Here, we identified the epimerization reactions and the responsible catalytic residues through observation of the reactions of FbaA and l-rhamnulose-1-phosphate aldolases (RhaD) variants with substituted catalytic residues using different substrates. Moreover, we obtained detailed potential epimerization reaction mechanism of FbaA and a general epimerization mechanism of the class II aldolases l-fuculose-1-phosphate aldolase, RhaD, and FbaA. Thus, class II aldolases can be used as 4-epimerases for the stereo-selective synthesis of valuable carbohydrates.
“…2). Although Escherichia coli FbaA did not epimerize FBP to TBP due to having a small cavity volume, it can epimerize the smaller substrate F6P to T6P 19 .Figure 2Bio-LC diagram and 31 P-NMR spectra for the reaction of F6P with FbaA. ( A ) Bio-LC diagrma of T6P biocoversion from F6P by FbaA.…”
Sugar 4-epimerization reactions are important for the production of rare sugars and their derivatives, which have various potential industrial applications. For example, the production of tagatose, a functional sweetener, from fructose by sugar 4-epimerization is currently constrained because a fructose 4-epimerase does not exist in nature. We found that class II d-fructose-1,6-bisphosphate aldolase (FbaA) catalyzed the 4-epimerization of d-fructose-6-phosphate (F6P) to d-tagatose-6-phosphate (T6P) based on the prediction via structural comparisons with epimerase and molecular docking and the identification of the condensed products of C3 sugars. In vivo, the 4-epimerization activity of FbaA is normally repressed. This can be explained by our results showing the catalytic efficiency of d-fructose-6-phosphate kinase for F6P phosphorylation was significantly higher than that of FbaA for F6P epimerization. Here, we identified the epimerization reactions and the responsible catalytic residues through observation of the reactions of FbaA and l-rhamnulose-1-phosphate aldolases (RhaD) variants with substituted catalytic residues using different substrates. Moreover, we obtained detailed potential epimerization reaction mechanism of FbaA and a general epimerization mechanism of the class II aldolases l-fuculose-1-phosphate aldolase, RhaD, and FbaA. Thus, class II aldolases can be used as 4-epimerases for the stereo-selective synthesis of valuable carbohydrates.
“…Unlike the active site loop within class II FBAs, the β6α6 loop containing residues 177–191, and previously termed the Z-loop, is almost always well-defined structurally. ,,,,,,,,− This loop forms part of the substrate pocket as well as contains a histidine residue involved in coordination with the active site Zn(II) ion . Between class IIb FBAs, such as HpFBA, whose structures have been reported, and SaFBA, HpFBA possess two additional residues within this region.…”
Section: Resultsmentioning
confidence: 99%
“…Previously, class II FBAs could be grouped into either IIa or IIb on the basis of their sequence homology and oligomeric state. ,,,, Genetic studies of several pathogenic bacteria in which the fba gene was removed resulted in a loss of viability for the organism. − As a result, over the past few years the potential benefit of using class II FBAs as targets for therapeutic development has spurred the elucidation of a number of class II FBA structures. ,,,,,, Taking this additional structural information into consideration, the differences within these classes become readily apparent. For instance among class IIa FBAs, MtFBA possesses several helices facilitating a tetrameric organization setting itself structurally apart from all other structurally characterized class IIa FBAs, such as the one from E. coli as well as a recent unreported protein data bank entry from Campylobacter jejuni (PDB Code: 3QM3).…”
Section: Discussionmentioning
confidence: 99%
“… 40 , 41 To date, only the structures of class IIb-i FBAs have been studied in regard to their enzymatic capacities. 20 , 42 − 45 Based on sequence composition, SaFBA appears to belong to the class IIb-iv of FBAs; however, no structure of either SaFBA, or a proposed class IIb-iv FBA, has yet been reported (Figure 1 ).…”
mentioning
confidence: 99%
“…Although class IIa FBAs were traditionally thought to be all dimers, recent findings have shown that there exist certain exceptions, such as the class IIa FBA from Mycobacterium tuberculosis (MtFBA), which forms a tetramer . Meanwhile, class IIb FBAs can range from dimers to tetramers to even octomers and tend to be shorter in amino acid length than their class IIa counterparts. ,,,, Furthermore, sequence alignments of class IIb FBAs suggest that this group might be divided into at least four additional subtypes (i-iv) although certain discrepancies exist within the purely sequence-based categorization. , To date, only the structures of class IIb-i FBAs have been studied in regard to their enzymatic capacities. ,− Based on sequence composition, SaFBA appears to belong to the class IIb-iv of FBAs; however, no structure of either SaFBA, or a proposed class IIb-iv FBA, has yet been reported (Figure ).…”
Staphylococcus aureus is one of the most common
nosocomial sources of soft-tissue and skin infections and has more
recently become prevalent in the community setting as well. Since
the use of penicillins to combat S. aureus infections
in the 1940s, the bacterium has been notorious for developing resistances
to antibiotics, such as methicillin-resistant Staphylococcus
aureus (MRSA). With the persistence of MRSA as well as many
other drug resistant bacteria and parasites, there is a growing need
to focus on new pharmacological targets. Recently, class II fructose
1,6-bisphosphate aldolases (FBAs) have garnered attention to fill
this role. Regrettably, scarce biochemical data and no structural
data are currently available for the class II FBA found in MRSA (SaFBA).
With the recent finding of a flexible active site zinc-binding loop
(Z-Loop) in class IIa FBAs and its potential for broad spectrum class
II FBA inhibition, the lack of information regarding this feature
of class IIb FBAs, such as SaFBA, has been limiting for further Z-loop
inhibitor development. Therefore, we elucidated the crystal structure
of SaFBA to 2.1 Å allowing for a more direct structural analysis
of SaFBA. Furthermore, we determined the KM for one of SaFBA’s substrates, fructose 1,6-bisphosphate,
as well as performed mode of inhibition studies for an inhibitor that
takes advantage of the Z-loop’s flexibility. Together the data
offers insight into a class IIb FBA from a pervasively drug resistant
bacterium and a comparison of Z-loops and other features between the
different subtypes of class II FBAs.
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