Kinetic characterization of annotated glycolytic enzymes present in cellulose-fermenting Clostridium thermocellum suggests different metabolic roles

Daley, Steve R.; Gallanosa, Patricia Mae; Sparling, Richard

doi:10.1186/s13068-023-02362-8

Biotechnology for Biofuels and Bioproducts

Table 4 Characterized Pfks from two of the three phylogenetically distinct but related families

From: Kinetic characterization of annotated glycolytic enzymes present in cellulose-fermenting Clostridium thermocellum suggests different metabolic roles

Organism	Phylum	\(V\mathrm{max}(\frac{\mathrm{\mu mol}}{\mathrm{mg min}})\)								K_m (mM)					Reference
Family	PfkA^l
Geobacillus stearothermophilus	Firmicutes, thermophile	–								F6P 0.033			ATP 0.07		[86]
Thermotoga maritima	Thermotogae, hyperthermophilic	F6P 464	ATP 432						GTP 294	F6P 0.437	ATP 0.009			GTP 1.36	[45]
Thermus thermophilus	Deinococcus-Thermus, extreme thermophile	–								F6P 0.025				ATP 0.08	[87]
Streptomyces coelicolor A3(2)	Actinobacteria, mesophile	F6P/ATP^a 165								F6P/ATP 1.0/0.4					[88]
Mycobacterium tuberculosis H37Rv	Actinobacteria, mesophile	F6P 20		ATP 19				T6P 30		F6P 0.4	ATP 1			T6P^J 0.43	[43]
Amycolatopsis methanolica	Actinobacteria, mesophilic or moderately thermophilic	F6P/ATP^m 167/180								F6P/ATP 6.0/0.6					[59]
Lactobacillus delbrueckii subsp. bulgaricus	Bacillota	F6P/ATP 90								F6P 0.3	ATP(pH 8.2) 0.2			ATP (pH 6.0)0.07	[58]
	Pfp^l
Thermotoga maritima	Thermotogae, hyperthermophilic	PP_i 203	PPP_i 249				Poly-P 319			PP_i 0.067		PPP_i 0.010		Poly-P 0.0038	[45]
Methylococcus capsulatus^c	Proteobacteria, thermotolerant	F6P 7.6	S7P 31				FBP 9.0			F6P 2.27		S7P 0.03		PP_i .0.027	[41]
Rhodospirillum rubrum	Proteobacteria, mesophile	F6P 20			FBP 24.2					F6P 0.38		FBP 0.02		PP_i 0.025	[89]
Xanthomonas campestris pv	g-Proteobacteria, mesophile	F6P 58			FBP 59					F6P .202		FBP 0.024		PP_i .041	[90]
Amycolatopsis methanolica	Actinobacteria, mesophilic or moderately thermophilic	F6P 58				FBP 59				F6P 0.4	FBP 0.025			PPi 0.2	[91]
Entamoeba histolytica	Amoebozoa	–								F6P 0.038	S7P 0.064			PPi .014	[40, 92]
Borrelia burgdorferi	Spirochaetes	–								F6P .109				PPI .015	[93]
PfkB
Mycobacterium tuberculosis H37Rv^b	Actinobacteria, mesophile	F6P 1.2			T6P 1.5					F6P 0.04		T6P^J 0.09			[43]
E. coli K12	Proteobacteria, mesophile	–			–					F6P 0.006		ATP 0.008			[50]
E. coli K12	Proteobacteria, mesophile	D-Ribose^e 81			ATP^f 122					d-Ribose^e 0.279		ATP 0.213			[48]
Staphylococcus aureus	Firmicutes	–			–					ATP 4.66		ATP^d 2.19			[94]
Thermus thermophilus	Deinococcota	–			–					KDG^g 0.32		KG 3.6			[95]
Sulfurisphaera tokodaii	Crenarchaeota	KDG 4.13			KG 13.26					KDG^h 0.027		KGⁱ 1.3			[96]
Saccharolobus solfataricus	Crenarchaeota	KDG 9.7								KDG^k(@ 70 °C) 0.14				ATP (@ 60 °C) 2.8	[97, 98]

^aMichaelis–Menten kinetics displayed with ATP(1 mM) and varying concentration of F6P up to 10 mM
^bThis Pfk B was also able to catalyze the gluconeogenic reaction with FBP, ADP, IDP and GDP as substrates
^cThe K_m of phosphate and diphosphate are 8.69 mM and 0.027 mM, respectively
^dIn the presence of 100 mM K⁺
^eRecombinant enzyme also able to utilize 2-deoxy-d-ribose, d-arabinose, d-xylose and d-fructose as substrate but less efficiently. K_m of d-ribose also decreases with the addition of pentavalent ions (phosphate, vanadate, arsenate), as well as increased V_max
^fGTP was also a phosphate donor
^g2-Keto-3-deoxygluconate (KDG) & 2-keto-d-gluconate (KG) were the substrates under investigation. Activity recorder as k_cat for each substrate was excluded from the table for comparative reasons with V_max
^hK_m of ATP with KDG is 0.036
ⁱK_m of ATP with KG is 0.11
^JK_0.5 rather than K_m, as kinetic data were fitted to a sigmoidal (Hill) response curve
^kK_m of KDG increases to 3.6 mM at 60 °C. 2-keto-3-deoxygalactonate is also a substrate, but affinity is twofold lower than KDG under similar conditions
^lPfkA and Pfp both belong to Family A PFK
^mV_max of PfkA when ATP concentration held constant at 1 mM while [F6P] varied, followed by V_max when [F6P] held constant at 10 mM while [ATP] varied

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