UDP-glucuronic acid (UDP-GlcA) 4-epimerase illustrates an important problem regarding enzyme catalysis: balancing conformational flexibility with precise positioning. The enzyme coordinates the C4oxidation of the substrate by NAD + and rotation of a decarboxylation-prone β-keto acid intermediate in the active site, enabling stereoinverting reduction of the keto group by NADH. We reveal the elusive rotational landscape of the 4-keto intermediate. Distortion of the sugar ring into boat conformations induces torsional mobility in the enzyme's binding pocket. The rotational endpoints show that the 4-keto sugar has an undistorted 4 C 1 chair conformation. The equatorially placed carboxylate group disfavors decarboxylation of the 4-keto sugar. Epimerase variants lead to decarboxylation upon removal of the binding interactions with the carboxylate group in the opposite rotational isomer of the substrate. Substitutions R185A/D convert the epimerase into UDP-xylose synthases that decarboxylate UDP-GlcA in stereospecific, configuration-retaining reactions.
Bifidobacterium
bifidum
lacto-
N
-biosidase (LnbB)
is a critical enzyme for the degradation
of human milk oligosaccharides in the gut microbiota of breast-fed
infants. Guided by recent crystal structures, we unveil its molecular
mechanism of catalysis using QM/MM metadynamics. We show that the
oligosaccharide substrate follows
1
S
3
/
1,4
B
→ [
4
E
]
‡
→
4
C
1
/
4
H
5
and
4
C
1
/
4
H
5
→ [
4
E
/
4
H
5
]
‡
→
1,4
B
conformational itineraries for the two
successive reaction steps, with reaction free energy barriers in agreement
with experiments. The simulations also identify a critical histidine
(His263) that switches between two orientations to modulate the p
K
a
of the acid/base residue, facilitating catalysis.
The reaction intermediate of LnbB is best depicted as an oxazolinium
ion, with a minor population of neutral oxazoline. The present study
sheds light on the processing of oligosaccharides of the early life
microbiota and will be useful for the engineering of LnbB and similar
glycosidases for biocatalysis.
Ratiometric fluorescent nanothermometers with near‐infrared emission play an important role in in vivo sensing since they can be used as intracellular thermal sensing probes with high spatial resolution and high sensitivity, to investigate cellular functions of interest in diagnosis and therapy, where current approaches are not effective. Herein, the temperature‐dependent fluorescence of organic nanoparticles is designed, synthesized, and studied based on the dual emission, generated by monomer and excimer species, of the tris(2,4,6‐trichlorophenyl)methyl radical (TTM) doping organic nanoparticles (TTMd‐ONPs), made of optically neutral tris(2,4,6‐trichlorophenyl)methane (TTM‐αH), acting as a matrix. The excimer emission intensity of TTMd‐ONPs decreases with increasing temperatures whereas the monomer emission is almost independent and can be used as an internal reference. TTMd‐ONPs show a great temperature sensitivity (3.4% K−1 at 328 K) and a wide temperature response at ambient conditions with excellent reversibility and high colloidal stability. In addition, TTMd‐ONPs are not cytotoxic and their ratiometric outputs are unaffected by changes in the environment. Individual TTMd‐ONPs are able to sense temperature changes at the nano‐microscale. In vivo thermometry experiments in Caenorhabditis elegans (C. elegans) worms show that TTMd‐ONPs can locally monitor internal body temperature changes with spatio‐temporal resolution and high sensitivity, offering multiple applications in the biological nanothermometry field.
UDP-Glucuronsäure(UDP-GlcA)-4-Epimerase repräsentiert eine wichtige Fragestellung in der Enzymkatalyse: die Balance zwischen konformativer Flexibilität und genauer Positionierung. Das Enzym koordiniert die C4-Oxidation des Substrats durch NAD + mit der Rotation eines leicht decarboxylierbaren β-Ketosäure-Intermediats im aktiven Zentrum zur Ermöglichung der stereoinvertierenden Reduktion der Ketogruppe durch NADH. Wir zeigen hier die nur schwer erfassbare Rotationskoordinate des 4-Ketointermediats. Distorsion des Zuckerrings in eine Boot-Konformation erzeugt torsionale Mobilität in der Bindungstasche des Enzyms. Die Endpunkte der Rotation zeigen den 4-Ketozucker in einer unverformten 4 C 1 -Sesselkonformation. Die äquatorial positionierte Carboxylatgruppe ist ungünstig für die 4-Ketozucker-Decarboxylierung. Varianten der Epimerase zeigen Decarboxylierung, wenn sie die Bindung mit der Carboxylatgruppe im entgegengesetzten Rotationsisomer des Substrats entfernen. R185A/D-Substitutionen wandeln die Epimerase in UDP-Xylose-Synthasen um, welche UDP-GlcA in stereospezifischen, konfigurationserhaltenden Reaktionen decarboxylieren.
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