Characterization of Fructose 1,6-Bisphosphatase and Sedoheptulose 1,7-Bisphosphatase from the Facultative Ribulose Monophosphate Cycle Methylotroph Bacillus methanolicus

Abstract

The genome of the facultative ribulose monophosphate (RuMP) cycle methylotroph Bacillus methanolicus encodes two bisphosphatases (GlpX), one on the chromosome (GlpX C ) and one on plasmid pBM19 (GlpX P ), which is required for methylotrophy. Both enzymes were purified from recombinant Escherichia coli and were shown to be active as fructose 1,6-bisphosphatases (FBPases). The FBPase-negative Corynebacterium glutamicum ⌬fbp mutant could be phenotypically complemented with glpX C and glpX P from B. methanolicus. GlpX P and GlpX C share similar functional properties, as they were found here to be active as homotetramers in vitro, activated by Mn 2؉ ions and inhibited by Li ؉ , but differed in terms of the kinetic parameters. GlpX C showed a much higher catalytic efficiency and a lower K m for fructose 1,6-bisphosphate (86.3 s ؊1 mM ؊1 and 14 ؎ 0.5 M, respectively) than GlpX P (8.8 s ؊1 mM ؊1 and 440 ؎ 7.6 M, respectively), indicating that GlpX C is the major FBPase of B. methanolicus. Both enzymes were tested for activity as sedoheptulose 1,7-bisphosphatase (SBPase), since a SBPase variant of the ribulose monophosphate cycle has been proposed for B. methanolicus. The substrate for the SBPase reaction, sedoheptulose 1,7-bisphosphate, could be synthesized in vitro by using both fructose 1,6-bisphosphate aldolase proteins from B. methanolicus. Evidence for activity as an SBPase could be obtained for GlpX P but not for GlpX C . Based on these in vitro data, GlpX P is a promiscuous SBPase/FBPase and might function in the RuMP cycle of B. methanolicus.

Synthetic methylotrophy: engineering the production of biofuels and chemicals based on the biology of aerobic methanol utilization

“…However, the plasmid-encoded enzyme also displayed activity toward sedoheptulose 1,7-bisphosphate whereas the chromosomal enzyme did not. This implies that the plasmid encoded fructose 1,6 bisphosphatase is the main enzyme of the RuMP regeneration pathway [25]. Similarly, the chromosomal fructose 1,6-bisphosphate aldolase exhibited higher catalytic efficiency for the aldol cleavage of fructose 1,6-bisphosphate, whereas the plasmid encoded version exhibited higher catalytic efficiency toward triose phosphates [26].…”

“…Second, several studies have compared the activities of both the plasmid and chromosomal RuMP-pathway regeneration genes and found that they possess somewhat different activities [24][25][26]. For example, the chromosomally encoded fructose 1,6 bisphosphatase had higher activity and a lower K m for fructose 1,6-bisphosphate compared with the plasmid-encoded copy.…”

Core pathways operating during methylotrophy of Bacillus methanolicus MGA3 and induction of a bacillithiol‐dependent detoxification pathway upon formaldehyde stress

“…The detection of high levels of hexulose‐6‐phosphate synthase (Hps) and 6‐phospho‐3‐hexuloisomerase (Phi) activity in cell‐free extracts of B. methanolicus made it likely that the RuMP pathway is employed for carbon assimilation (Dijkhuizen et al ., ; Arfman et al ., ). Genomic information together with transcriptome data, proteome data and enzymatic studies suggested that the fructose‐bisphosphate aldolase/sedoheptulose bisphosphatase variant of the RuMP cycle is used (Heggeset et al ., ; Stolzenberger et al ., 2013a,b; Müller et al ., ) (Fig. ).…”

“…For growth on methanol, transcriptome and proteome data have demonstrated that only the plasmid‐encoded versions of the genes are upregulated (Heggeset et al ., ; Müller et al ., ). The reason for the gene duplication is not known, but characterization of some of the enzymes indicated that the chromosomal‐ and plasmid‐encoded enzymes differ in their biochemical properties (Stolzenberger et al ., 2013a,b).…”

Characterization of two transketolases encoded on the chromosome and the plasmid pBM19 of the facultative ribulose monophosphate cycle methylotroph Bacillus methanolicus

“…Enzymes: MDH ,  methanol dehydrogenase (EC 1.1.1.244); HPS ,  3-hexulose-6-phosphate synthase (EC 4.1.2.43); PHI ,  6-phospho-3-hexuloisomerase (EC 5.3.1.27); PFK ,  6-phosphofructokinase, (EC 2.7.1.11); FBA ,  fructose-bisphosphate aldolase (EC 4.1.2.13); TKT ,  transketolase (EC 2.2.1.1); GlpX ,  fructose-bisphosphatase (EC 3.1.3.1); TA ,  transaldolase (EC 2.2.1.2); RPE , ribulose- phosphate 3-epimerase (EC 5.1.3.1); RPI ,  ribose-5-phosphate isomerase (EC 5.3.1.6); Metabolites: H6-P, 3-hexulose 6-phosphate; F6-P, fructose-6-phosphate; FBP, fructose-1,6-bisphosphate; GAP, glyceraldehyde 3-phosphate; DHAP, dihydroxyacetone phosphate; E4-P, erythrose 4-phosphate; SBP, sedoheptulose 1,7-bisphosphate; S7-P, sedoheptulose-7-phosphate; Ri5-P, ribose 5-phosphate; X5P, xylulose 5-phosphate; Ru5P, ribulose 5-phosphate; The reactions are described in detail in the text. Adapted from [28]. …”