several alcohols, being widely available from various aerobically grown sources,
and not requiring the use of external cofactors for catalysis [15].
ChOx (Fig. 1c) is a homodimeric enzyme, like GOx, but the FAD cofactor is
covalently bound to the respective monomer [18, 19]. ChOx catalyzes the
four-electron oxidation of choline to glycine betaine, producing betaine aldehyde as
an intermediate [18]. The enzyme has been applied in the development of
biosensing devices for the indirect determination of acetylcholine, a major excitatory neurotransmitter implied in several neurological disorders [20]. Through the
hydrolytic activity of acetylcholine esterase, the neurotransmitter is converted into
choline, which can then be oxidized by ChOx with the concomitant production of
electroactive H 2 O 2 under aerobic conditions [20, 21]. The bacterial, dimeric, and
FAD-dependent GluOx (Fig. 1d) has also been employed in the development of
sensing devices for neurotransmitter detection, by catalyzing the oxidative deamination of glutamate to 2-ketoglutarate [22, 23]. The former is the principal excitatory neurotransmitter in the central nervous system, with excessive release into the
cerebral extracellular space inducing neurotoxicity, and thus being implicated in
several neurological disorders [23].
The monomeric ChOD catalyzes the oxidation and isomerization of cholesterol
to cholest-4-en-3-one [24]. It is uniquely produced by several bacteria (e.g.,
Arthobacter, Mycobacterium, Streptomyces, Rhodococcus, Brevibacterium) that are
capable of using cholesterol as a carbon and energy source [24–26]. The enzyme
exists in two structurally diverse types that catalyze the same reactions: type I
presents a non-covalently bound FAD cofactor and belongs to the GMC superfamily, found in Streptomyces (Fig. 1e) and Rhodococcus; type II contains a
covalently bound FAD and is found in Brevibacterium and Burkholderia [26, 27].
Regarding clinical applications, ChOD has been used as an analytical probe in the
determination of cholesterol in physiological samples (plasma, serum, gall stones,
bile) [25, 28].
The multimeric LOx (Fig. 1f) catalyzes the conversion of a-hydroxy acids (e.g.,
lactate) to a-keto-acids (e.g., pyruvate), via a ping-pong mechanism [29]. The
enzyme from the bacterial source Aerococcus viridans crystallizes as two tightly
packed tetramers, with each one forming a biologically active unit [29, 30]. Each
subunit contains one FMN cofactor deeply buried in the active site, as opposed to a
FAD prosthetic group observed in other flavin-dependent oxidases [29, 30]. The
substrate lactate is the key metabolite of the anaerobic glycolytic pathway, so its
concentration in blood is an indirect biomarker of anaerobic glucose breakdown and
cellular fatigue, being widely used in the clinical diagnostics for assessing patient
health conditions and continuous surveillance in surgery, sports medicine,
shock/trauma and food industry [31, 32]. Therefore, LOx has been applied in the
development of lactate analytical devices, due to the combined simplicity of the
enzymatic reaction and sensor design fabrication [32].
XOD differs from the aforementioned oxidases in respect to the cofactors present
in the active site. The mammalian (human and bovine) enzyme is a homodimer
Fig. 1g), with each monomer containing a molybdopterin cofactor, two spectroscopically distinct Fe–S clusters, and one FAD [33, 34]. The catalytic oxidation of
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and not requiring the use of external cofactors for catalysis [15].
ChOx (Fig. 1c) is a homodimeric enzyme, like GOx, but the FAD cofactor is
covalently bound to the respective monomer [18, 19]. ChOx catalyzes the
four-electron oxidation of choline to glycine betaine, producing betaine aldehyde as
an intermediate [18]. The enzyme has been applied in the development of
biosensing devices for the indirect determination of acetylcholine, a major excitatory neurotransmitter implied in several neurological disorders [20]. Through the
hydrolytic activity of acetylcholine esterase, the neurotransmitter is converted into
choline, which can then be oxidized by ChOx with the concomitant production of
electroactive H 2 O 2 under aerobic conditions [20, 21]. The bacterial, dimeric, and
FAD-dependent GluOx (Fig. 1d) has also been employed in the development of
sensing devices for neurotransmitter detection, by catalyzing the oxidative deamination of glutamate to 2-ketoglutarate [22, 23]. The former is the principal excitatory neurotransmitter in the central nervous system, with excessive release into the
cerebral extracellular space inducing neurotoxicity, and thus being implicated in
several neurological disorders [23].
The monomeric ChOD catalyzes the oxidation and isomerization of cholesterol
to cholest-4-en-3-one [24]. It is uniquely produced by several bacteria (e.g.,
Arthobacter, Mycobacterium, Streptomyces, Rhodococcus, Brevibacterium) that are
capable of using cholesterol as a carbon and energy source [24–26]. The enzyme
exists in two structurally diverse types that catalyze the same reactions: type I
presents a non-covalently bound FAD cofactor and belongs to the GMC superfamily, found in Streptomyces (Fig. 1e) and Rhodococcus; type II contains a
covalently bound FAD and is found in Brevibacterium and Burkholderia [26, 27].
Regarding clinical applications, ChOD has been used as an analytical probe in the
determination of cholesterol in physiological samples (plasma, serum, gall stones,
bile) [25, 28].
The multimeric LOx (Fig. 1f) catalyzes the conversion of a-hydroxy acids (e.g.,
lactate) to a-keto-acids (e.g., pyruvate), via a ping-pong mechanism [29]. The
enzyme from the bacterial source Aerococcus viridans crystallizes as two tightly
packed tetramers, with each one forming a biologically active unit [29, 30]. Each
subunit contains one FMN cofactor deeply buried in the active site, as opposed to a
FAD prosthetic group observed in other flavin-dependent oxidases [29, 30]. The
substrate lactate is the key metabolite of the anaerobic glycolytic pathway, so its
concentration in blood is an indirect biomarker of anaerobic glucose breakdown and
cellular fatigue, being widely used in the clinical diagnostics for assessing patient
health conditions and continuous surveillance in surgery, sports medicine,
shock/trauma and food industry [31, 32]. Therefore, LOx has been applied in the
development of lactate analytical devices, due to the combined simplicity of the
enzymatic reaction and sensor design fabrication [32].
XOD differs from the aforementioned oxidases in respect to the cofactors present
in the active site. The mammalian (human and bovine) enzyme is a homodimer
Fig. 1g), with each monomer containing a molybdopterin cofactor, two spectroscopically distinct Fe–S clusters, and one FAD [33, 34]. The catalytic oxidation of
312
T. Monteiro et al.