Nucleophilic Participation of Reduced Flavin Coenzyme in Mechanism of UDP-galactopyranose Mutase

Sun, He; Ruszczycky, Mark W.; Chang, Wei‐chen; Thibodeaux, Christopher J.; Liu, Hung Wen

doi:10.1074/jbc.m111.312538

Cited by 36 publications

(49 citation statements)

References 33 publications

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“…Recently, flavin analogues were used to determine kinetic linear free energy relationship of k cat to the nucleophilicity of the N5 of the flavin. The results show a slope, ρ of -2.4, which is consistent with the direct attack of the N5 of the flavin to the UDP-Galp [77]. …”

Section: Udp-galactopyranose Mutasesupporting

confidence: 73%

Noncanonical Reactions of Flavoenzymes

Sobrado

2012

IJMS

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Enzymes containing flavin cofactors are predominantly involved in redox reactions in numerous cellular processes where the protein environment modulates the chemical reactivity of the flavin to either transfer one or two electrons. Some flavoenzymes catalyze reactions with no net redox change. In these reactions, the protein environment modulates the reactivity of the flavin to perform novel chemistries. Recent mechanistic and structural data supporting novel flavin functionalities in reactions catalyzed by chorismate synthase, type II isopentenyl diphosphate isomerase, UDP-galactopyranose mutase, and alkyl-dihydroxyacetonephosphate synthase are presented in this review. In these enzymes, the flavin plays either a direct role in acid/base reactions or as a nucleophile or electrophile. In addition, the flavin cofactor is proposed to function as a “molecular scaffold” in the formation of UDP-galactofuranose and alkyl-dihydroxyacetonephosphate by forming a covalent adduct with reaction intermediates.

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Section: Udp-galactopyranose Mutasesupporting

confidence: 73%

Noncanonical Reactions of Flavoenzymes

Sobrado

2012

IJMS

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“…Recently, Liu and coworkers probed the mechanism using flavin analogs with different nucleophilicities at the N5 FAD . The kinetic linear free energy relationship resulted in a slope of ρ= −2.45, consistent with a direct attack of the N5 FAD in an S N 2-type mechanism [33]. In addition, a flavin-iminium intermediate was observed during time-resolved spectroscopy of the reaction of UDP-Gal f with reduced T. cruzi UGM (TcUGM) without the formation of a flavin semiquinone intermediate, inconsistent with a single electron transfer mechanism [30].…”

Section: Chemical Mechanisms Of Ugmmentioning

confidence: 75%

Structure, mechanism, and dynamics of UDP-galactopyranose mutase

Tanner

Boechi

McCammon

et al. 2014

Archives of Biochemistry and Biophysics

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The flavoenzyme UDP-galactopyranose mutase (UGM) is a key enzyme in galactofuranose biosynthesis. The enzyme catalyzes the 6-to-5 ring contraction of UDP-galactopyranose to UDP-galactofuranose. Galactofuranose is absent in humans yet is an essential component of bacterial and fungal cell walls and a cell surface virulence factor in protozoan parasites. Thus, inhibition of galactofuranose biosynthesis is a valid strategy for developing new antimicrobials. UGM is an excellent target in this effort because the product of the UGM reaction represents the first appearance of galactofuranose in the biosynthetic pathway. The UGM reaction is redox neutral, which is atypical for flavoenzymes, motivating intense examination of the chemical mechanism and structural features that tune the flavin for its unique role in catalysis. These studies show that the flavin functions as nucleophile, forming a flavin-sugar adduct that facilitates galactose-ring opening and contraction. The 3-dimensional fold is novel and conserved among all UGMs, however the larger eukaryotic enzymes have additional secondary structure elements that lead to significant differences in quaternary structure, substrate conformation, and conformational flexibility. Here we present a comprehensive review of UGM three-dimensional structure, provide an update on recent developments in understanding the mechanism of the enzyme, and summarize computational studies of active site flexibility.

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“…The prevailing mechanism is an S N 2-type displacement in which the N5 atom of the reduced FAD functions as the nucleophile that attacks the anomeric carbon of galactose to form a covalent intermediate and displace UDP (38, 39). This mechanism was recently validated for TcUGM (26).…”

Section: Discussionmentioning

confidence: 99%

Crystal Structures of Trypanosoma cruzi UDP-Galactopyranose Mutase Implicate Flexibility of the Histidine Loop in Enzyme Activation

et al. 2012

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Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. Here we report crystal structures of the galactofuranose biosynthetic enzyme UDP-galactopyranose mutase (UGM) from T. cruzi, which are the first structures of this enzyme from a protozoan parasite. UGM is an attractive target for drug design because galactofuranose is absent in humans but is an essential component of key glycoproteins and glycolipids in trypanosomatids. Analysis of the enzyme-UDP noncovalent interactions and sequence alignments suggests that substrate recognition is exquisitely conserved among eukaryotic UGMs and distinct from that of bacterial UGMs. This observation has implications for inhibitor design. Activation of the enzyme via reduction of the FAD induces profound conformational changes, including a 2.3-Å movement of the histidine loop (Gly60-Gly61-His62), rotation and protonation of the imidazole of His62, and cooperative movement of residues located on the si face of the FAD. Interestingly, these changes are substantially different from those described for Aspergillus fumigatus UGM, which is 45 % identical to T. cruzi UGM. The importance of Gly61 and His62 for enzymatic activity was studied with the site-directed mutant enzymes G61A, G61P, and H62A. These mutations lower the catalytic efficiency by factors of 10–50, primarily by decreasing kcat. Considered together, the structural, kinetic, and sequence data suggest that the middle Gly of the histidine loop imparts flexibility that is essential for activation of eukaryotic UGMs. Our results provide new information about UGM biochemistry and suggest a unified strategy for designing inhibitors of UGMs from the eukaryotic pathogens.

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Nucleophilic Participation of Reduced Flavin Coenzyme in Mechanism of UDP-galactopyranose Mutase

Cited by 36 publications

References 33 publications

Noncanonical Reactions of Flavoenzymes

Noncanonical Reactions of Flavoenzymes

Structure, mechanism, and dynamics of UDP-galactopyranose mutase

Crystal Structures of Trypanosoma cruzi UDP-Galactopyranose Mutase Implicate Flexibility of the Histidine Loop in Enzyme Activation

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