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

Abstract

BackgroundTransketolase (TKT) is a key enzyme of the pentose phosphate pathway (PPP), the Calvin cycle and the ribulose monophosphate (RuMP) cycle. Bacillus methanolicus is a facultative RuMP pathway methylotroph. B. methanolicus MGA3 harbors two genes putatively coding for TKTs; one located on the chromosome (tkt C ) and one located on the natural occurring plasmid pBM19 (tkt P ).ResultsBoth enzymes were produced in recombinant Escherichia coli, purified and shown to share similar biochemical parameters in vitro. They were found to be active as homotetramers and require thiamine pyrophosphate for catalytic activity. The inactive apoform of the TKTs, yielded by dialysis against buffer containing 10 mM EDTA, could be reconstituted most efficiently with Mn2+ and Mg2+. Both TKTs were thermo stable at physiological temperature (up to 65°C) with the highest activity at neutral pH. Ni2+, ATP and ADP significantly inhibited activity of both TKTs. Unlike the recently characterized RuMP pathway enzymes fructose 1,6-bisphosphate aldolase (FBA) and fructose 1,6-bisphosphatase/sedoheptulose 1,7-bisphosphatase (FBPase/SBPase) from B. methanolicus MGA3, both TKTs exhibited similar kinetic parameters although they only share 76% identical amino acids. The kinetic parameters were determined for the reaction with the substrates xylulose 5-phosphate (TKTC: kcat/KM: 264 s-1 mM-1; TKTP: kcat/KM: 231 s-1 mM) and ribulose 5-phosphate (TKTC: kcat/KM: 109 s-1 mM; TKTP: kcat/KM: 84 s-1 mM) as well as for the reaction with the substrates glyceraldehyde 3-phosphate (TKTC: kcat/KM: 108 s-1 mM; TKTP: kcat/KM: 71 s-1 mM) and fructose 6-phosphate (TKTC kcat/KM: 115 s-1 mM; TKTP: kcat/KM: 448 s-1 mM).ConclusionsBased on the kinetic parameters no major TKT of B. methanolicus could be determined. Increased expression of tkt P , but not of tkt C during growth with methanol [J Bacteriol 188:3063–3072, 2006] argues for TKTP being the major TKT relevant in the RuMP pathway. Neither TKT exhibited activity as dihydroxyacetone synthase, as found in methylotrophic yeast, or as the evolutionary related 1-deoxyxylulose-5-phosphate synthase. The biological significance of the two TKTs for B. methanolicus methylotrophy is discussed.

In vitro activation of NAD‐dependent alcohol dehydrogenases by Nudix hydrolases is more widespread than assumed

“…B. methanolicus MGA3 Mdh and Mdh2 activities were previously shown to be increased by Act under alkaline pH conditions [5]. However, because neutral intracellular pH can be assumed for B. methanolicus [18,19], we decided to additionally study the enzymes at pH 7.4 to represent more physiological conditions. The enzyme activities of Mdh and Mdh2 were determined using methanol as a substrate at pH 9.0 and pH 7.4.…”

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

“…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.…”

Salmonella enterica serovar Typhimurium has three transketolase enzymes contributing to the pentose phosphate pathway

“…5C). In other species for which transketolase kinetic constants have been determined, k cat values can be similar to 10-fold lower, as is the case with Saccharomyces cerevisiae, or more than 3 orders of magnitude lower, as in Bacillus methanolicus (42,43). These observations raise questions about the minimal level of transketolase activity required for growth and why WT S. Typhimurium utilizes three transketolase isoenzymes that exhibit high levels of transketolase activity.…”