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

Abstract

Background: Transketolase (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). Results: Both 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 Mn§ssup§2+§esup§ and Mg§ssup§2+§esup§. Both TKTs were thermo stable at physiological temperature (up to 65°C) with the highest activity at neutral pH. Ni§ssup§2+§esup§, 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 (TKT§ssup§C§esup§: k§ssub§cat§esub§/K§ssub§M§esub§: 264 s§ssup§-1§esup§ mM§ssup§-1§esup§; TKT§ssup§P§esup§: k§ssub§cat§esub§/ K§ssub§M§esub§: 231 s§ssup§-1§esup§ mM) and ribulose 5-phosphate (TKT§ssup§C§esup§: k§ssub§cat§esub§/K§ssub§M§esub§: 109 s§ssup§-1§esup§ mM; TKT§ssup§P§esup§: k§ssub§cat§esub§/K§ssub§M§esub§: 84 s§ssup§-1§esup§ mM) as well as for the reaction with the substrates glyceraldehyde 3-phosphate (TKT§ssup§C§esup§: k§ssub§cat§esub§/K§ssub§M§esub§: 108 s§ssup§-1§esup§ mM; TKT§ssup§P§esup§: k§ssub§cat§esub§/K§ssub§M§esub§: 71 s§ssup§-1§esup§ mM) and fructose 6-phosphate (TKT§ssup§C§esup§ k§ssub§cat§esub§/ K§ssub§M§esub§: 115 s§ssup§-1§esup§ mM; TKT§ssup§P§esup§: k§ssub§cat§esub§/ K§ssub§M§esub§: 448 s§ssup§-1§esup§ mM). Conclusions: Based 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 TKT§ssup§P§esup§ 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. © 2014 Markert et al.; licensee BioMed Central Ltd.