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Phosphoketolase - A mechanistic update

Fabienne Libuda
Doctoral thesis
Date of Examination: 2017-11-30
Date of issue: 2018-08-22
Advisor: Tittmann, Kai Prof. Dr.
Referee: Ficner, Ralf Prof. Dr.
Referee: Mata, Ricardo Prof. Dr.

Persistent Address: http://hdl.handle.net/11858/00-1735-0000-002E-E48C-9

 

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Abstract

English

The thiamine diphosphate (ThDP)-dependent enzyme phosphoketolase catalyzes the phosphorolytic cleavage of fructose-6-phosphate and/or xylulose 5-phosphate under the generation of the high-energy metabolite acetyl phosphate, as such playing a key role in the metabolism of lactic acid bacteria and bifidobacteria. Phosphoketolase shows remarkable similarity to the prominent ThDP-dependent enzyme transketolase and catalyzes as only known ThDP-dependent enzyme a dehydration of its central alpha-carbanion/enamine intermediate. Thanks to a small number of structural and functional studies, the phosphoketolase catalytic mechanism is fairly well characterized. Dehydration of the alpha-carbanion/enamine cofactor adduct (alpha,beta-dihydroxyethyl)-thiamine diphosphate (DHEThDP) produces an enol-acetyl-ThDP intermediate that needs to undergo tautomerization to the corresponding keto-tautomer before a nucleophilic attack from the second substrate phosphate leads to product formation. In this thesis, a detailed analysis of the phosphoketolase reaction by kinetic and spectroscopic techniques was carried out with special emphasis on the mechanism of acetyl-thiamine diphosphate (AcThDP) tautomerization and the underlying chemical state of the post-dehydration intermediate as well as potential carboligase side reactivity of the enzyme. The obtained results provide new insights into the phosphoketolase reaction mechanism that suggest a mechanistic update for the enol-keto-tautomerization of the post-dehydration intermediate AcThDP, give new information on the similarities and differences of phosphoketolase and transketolase and led to the discovery of a novel ThDP-catalyzed C-C bond forming side reaction. Identification of a distinct UV-Vis absorbance signal associated to the phosphoketolase reaction intermediate AcThDP provided the basis for a transient kinetic analysis of individual steps of the phosphoketolase reaction. Additionally, a direct steady-state assay was established that allowed determination of kinetic parameters for the phosphoketolase main reaction as well as for the off-pathway hydrolysis of AcThDP to acetate that occurs in absence of the acyl-acceptor substrate phosphate. The detailed kinetic examination revealed that the rate of enol-keto tautomerization of AcThDP is increased by the presence of the acyl-acceptor substrate phosphate. Together with results from spectroscopic and computational analysis of the phosphoketolase reaction, this suggest a mechanism in which phosphate acts as substrate catalyst in the tautomerization from enol- to keto-AcThDP. Direct catalytic involvement of phosphate provides coupling of the final acyl-transfer to the preceding tautomerization which avoids formation of the hydrolysis-susceptible keto-AcThDP intermediate in the absence of an acceptor substrate. The proposed mechanism for AcThDP tautomerization implies formation of an enolate-AcThDP intermediate. Presence of this intermediate could be confirmed by trapping the enolate in a carboligation reaction with formaldehyde. The side reaction between phosphoketolase bound enolate-AcThDP and the aldehyde acceptor represents a novel type of ThDP-catalyzed C-C bond forming reaction as it not originates from the central alpha-carbanion/enamine intermediate. Generation of a "transketolase-like" phosphoketolase variant could not induce transketolase activity, but showed the importance of three phosphoketolase-specific active side residues for the phosphoketolase reaction mechanism.
Keywords: Phosphoketolase; Thiamin diphosphate; Biocatalysis; ThDP-dependent enzymes; Transketolase; Enzyme catalysis; Carboligation; Catalytic mechanism; AcThDP; Enolate-Intermediate
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