Article Figures & Data
Figures
- Fig. 1.
Schematic representation of the pathways for glucose and methanol metabolism in A. methanolica. GK, glucose kinase; FK, fructose kinase; PGI, phosphoglucose isomerase; TA, transaldolase; TK, transketolase; Ri5Pepi, ribulose-5-phosphate epimerase; Ri5Piso, ribulose-5-phosphate isomerase; TPI, triose phosphate isomerase, GAPdh, glyceraldehyde-3-phosphate dehydrogenase; S7P, sedoheptulose-7-phosphate; E4P, erythrose-4-phosphate; X5P, xylulose-5-phosphate; Ru5P, ribulose-5-phosphate; H6P, hexulose-6-phosphate; 1,3PG, 1,3-diphosphoglycerate; HPS, hexulose phosphate synthase; HPI, hexulose phosphate isomerase. The RuMP cycle enzymes specifically involved in the FBP aldolase cleavage variant (ATP-PFK and FBP aldolase) are shown by dashed lines.
- Fig. 2.
Profiles of ATP- and PPi-dependent PFK activities in cell extracts of A. methanolica grown in carbon- and energy source-limited continuous culture on the substrates glucose, formaldehyde, and methanol at a growth rate ofD (dilution rate) = 0.05 h−1. Days 0 to 6, 50 mM methanol; days 6 to 10, 50 mM methanol plus 5 mM formaldehyde; days 10 to 14, 50 mM methanol plus 12 mM formaldehyde; days 14 to 22, 50 mM formaldehyde; day 22 to end, 5 mM glucose. ●, ATP-PFK; ○, PPi-PFK.
- Fig. 3.
Kinetics of ATP-PFK from A. methanolicaand effect of PPi on ATP-PFK activity. (A) ATP-PFK activity versus ATP concentration at a fixed F-6-P concentration (10 mM). •, 0 mM PPi; ●, 1 mM PPi; ▾, 2 mM PPi; ▴, 4 mM PPi. (B) ATP-PFK activity versus F-6-P concentration at fixed ATP concentrations. ○, 0 mM PPi; ▵, 2 mM PPi; ■, 4 mM PPi. Kinetic parameters are given in Table 4.
- Fig. 4.
Clustal W (36) multiple alignment of the deduced amino acid sequence of the ATP-PFK from A. methanolica with the sequences of the PFKs from S. coelicolor A3(2) (National Center for Biotechnology Information [NCBI] accession number 008333), E. coli (NCBI accession number KIECFA), T. pallidum (NCBI accession number A71366 ), and T. brucei (NCBI accession numberAAC47836 ) and the PPi-PFK from A. methanolica (NCBI accession numberQ59126 ). The percent similarity between the ATP-PFK from A. methanolica and each of the other PFKs is indicated in parentheses. A and F indicate residues (solid boxes) involved in binding of ATP and F-6-P, respectively, in the E. coliATP-PFK enzyme (34). *, position with a fully conserved amino acid residue; :, position with a fully conserved strong group (STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, and FYW); ., position with a fully conserved weaker group (CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, FVLIM, and HFY).
- Fig. 5.
Unrooted phylogenetic tree of PFK proteins. The tree is based on distance analysis (neighbor-joining method) of selected available sequences of ATP-PFK and PPi-PFK proteins from the following sources in the National Center for Biotechnology Information database (accession numbers are given in parentheses):B. burgdorferi (D70102 ), Entamoeba histolytica (S68243 ), Giardia lamblia (S52081 ),Solanum tuberosum beta subunit (P21343 ), S. tuberosum alpha subunit (A36094 ), Arabidopsis thaliana (AC015450 .3), Thermotoga maritima(PPi-PFK) (G72396 ), Trichomonas vaginalis(AAD13344 ), Naegleria fowleri (S54978 ), T. brucei (AAC47836 ), A. methanolica (ATP-PFK) (AF298119 ), B. burgdorferi (putative PFK) (F70190 ), T. pallidum (A71366 ), Propionibacterium shermanii (A41169 ), Mycoplasma genitalium(G64223 ), Thermus thermophilus (P21777 ), T. maritima (ATP-PFK) (C72406 ), Aquifex aeolicus(O67605 ), E. coli (KIECFA), Lactococcus lactis (JN0614), Lactobacillus delbrueckii(A48663 ), Bacillus subtilis (A69675 ),Thermoproteus tenax (CAA74985 ), Dictyoglomus thermophilum (AF268276 .1), S. coelicolor A3(2) (PFK1) (OO8333), A. methanolica (PPi-PFK) (Q59126 ), S. coelicolor A3(2) (putative PFK) PFK2 (AL138978 .1), S. coelicolor A3(2) (putative PFK) PFK3 (AL391017 .1), M. tuberculosis (O53257 ), M. leprae (O33106 ), Saccharomyces cerevisae alpha subunit (NP011756.1), S. cerevisae beta subunit (NP013932), rat (A53047 ), rabbit (P00125128), and human muscle (227448). Bootstrap values, based on 100 replicates, are indicated at the branch points. The asterisks indicate putative PFKs.
- Fig. 6.
31P-NMR spectra of perchloric acid extracts of A. methanolica cells grown on mineral medium containing 60 mM methanol (spectrum A) or 10 mM glucose (spectrum B). Resonance assignments are indicated above each peak: ADP, ATP, UDP, UTP, UDP-hex (uridine diphosphohexoses, such as UDP-N-acetylglucosamine), PPi, Pi (inorganic phosphate), and poly-P (polyphosphate).
Tables
- Table 1.
Bacterial strains and plasmids used in this study
Strain or plasmid Relevant genotype Source or reference A. methanolicaWVI pMEA300-free derivative strain of A. methanolica NCBI 11946 42 E. coliDH5α supE44 ΔlacU169(φ80lacZΔM15)hsdR17 recA1 endA1 gyrA96 thi-1 relA1 Bethesda Research Laboratories Plasmids PDA71 Rhodococcus-E. colishuttle vector; cat ecoRI bla 10 pBluescript KS(+) Ampr; phagemid derived from pUC18;lacZ Stratagene PAM101 5-kb BamHI DNA fragment containing pfk ligated in BglII-digested pDA71 This study - Table 2.
Enzyme activities in A. methanolica WVI cells grown in batch culture on different substrates
Substrate Specific activity (mU · mg−1) PPi-PFK ATP-PFK Glucose (10 mM) 180 0 Fructose (10 mM) 120 0 Glycerol (20 mM) 100 0 Acetate (20 mM) 20 0 Gluconate (10 mM) 30 0 Succinate (10 mM) 20 0 Phenylalanine (10 mM) 30 0 Methanol (60 mM) 20 120 Formaldehydea (50 mM) 10 220 Betaine (20 mM) 10 180 ↵a Data from formaldehyde-limited continuous cultures in steady-state at a growth rate ofD (dilution rate) = 0.05 h−1.
- Table 3.
Purification of ATP-PFK from methanol-grown cells ofA. methanolica WVI
Step Procedure Protein (mg) Total activity (U) Sp act (U · mg−1) Purification (fold) Yield (%) 1 Crude extract 160 20.8 0.13 1 100 2 (NH4)2SO4(30%) 80 13.6 0.17 1.3 65 3 Levafix blue 0.2 10 50 385 48 4 Resource Q 0.08 7.2 90 692 35 5 Phenyl-Superose 0.03 4.8 160 1,230 23 - Table 4.
Kinetics of the ATP-PFK enzyme from A. methanolica WVI and effects of PPi on affinities for substrates F-6-P and ATP
Substrate Vmaxapp(U · mg−1) Kapp(mM) K50(mM) n PPi (mM) ATPa 180 ± 4.0 0.6 ± 0.1 Absent 130 ± 5.0 0.9 ± 0.07 1 110 ± 5.0 1.0 ± 0.1 2 2 60 ± 4.0 1.5 ± 0.1 2 4 F-6-Pb 167 ± 3.0 6.0 ± 0.17 2 Absent 100 ± 1.2 10 ± 1.0 2 2 80 ± 5 10 ± 0.8 2 4
