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

Benno Markert,Volker F Wendisch,Trygve Brautaset,Jessica Stolzenberger

doi:10.1186/1471-2180-14-7

Benno Markert, Volker F Wendisch + Show 2 more

Open Access

https://doi.org/10.1186/1471-2180-14-7

Copy DOI

Journal: BMC Microbiology	Publication Date: Jan 9, 2014
Citations: 70	License type: cc-by

Affiliation: Bielefeld University, SINTEF

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.

Highlights

Transketolase (TKT) is a key enzyme of the pentose phosphate pathway (PPP), the Calvin cycle and the ribulose monophosphate (RuMP) cycle
TKT is a key enzyme of the non-oxidative branch of the pentose phosphate pathway (PPP), the Calvin cycle and the ribulose monophosphate (RuMP) cycle
In the TA variant, sedoheptulose 7-phosphate (S7-P) is directly generated from fructose 6-phosphate (F6-P) and erythrose 4-phosphate (E4-P) by a TA activity, while the Sedoheptulose 1 (SBPase) variant requires the activity of a sedoheptulose 1,7-bisphosphate aldolase (SBA) to generate sedoheptulose 1,7-bisphosphate (SBP) and an SBPase activity for the further conversion of SBP to S7-P. That both FBAs from B. methanolicus are promiscuous enzymes active as SBA, while only the plasmid encoded GlpXP was active as Fructose 1 (FBPase) and SBPase, which indicates that the SBPase variant of the RuMP pathway might operate in this organism [28]

Summary

Introduction

Transketolase (TKT) is a key enzyme of the pentose phosphate pathway (PPP), the Calvin cycle and the ribulose monophosphate (RuMP) cycle. TKT is a key enzyme of the non-oxidative branch of the pentose phosphate pathway (PPP), the Calvin cycle and the ribulose monophosphate (RuMP) cycle In these metabolic pathways, two ketol group transfers are relevant, the interconversion of xylulose 5-phosphate (X5-P) and ribose 5-phosphate (R5-P) to sedoheptulose 7-phosphate (S7-P) and glyceraldehyde phosphate (GAP) and the interconversion of GAP and fructose 6-phosphate (F6-P) to erythrose 4-phosphate (E4-P) and X5-P [5]. Two ketol group transfers are relevant, the interconversion of xylulose 5-phosphate (X5-P) and ribose 5-phosphate (R5-P) to sedoheptulose 7-phosphate (S7-P) and glyceraldehyde phosphate (GAP) and the interconversion of GAP and fructose 6-phosphate (F6-P) to erythrose 4-phosphate (E4-P) and X5-P [5] These substrates of TKT are important as precursors e.g. for nucleotide biosynthesis (R5-P), biosynthesis of aromatic amino acids (E4-P) and for cell wall biosynthesis in Gram-negative bacteria (S7-P). They are intermediates of central pathways of carbon metabolism e.g. glycolysis (F6-P and GAP) and the Calvin and RuMP pathways [6]

Objectives

Methods

Results

Conclusion