B. Lakavath, et al.
Analytical Biochemistry 600 (2020) 113749
indicating retention of protein secondary structure. Activity assays in-
dicated that blue light inactivation occurs in the presence and absence
of oxygen. Inactivation also occurs in organic (Tris-HCl) as well as in-
organic (potassium phosphate) buffers. Photoinactivation is therefore
an intrinsic property of FAP that is not dependent on other assay re-
agents/oxygen, and most likely leads to localised modification of pro-
tein structure in, or close to, the enzyme active site. To retain catalytic
activity in purified samples, therefore, we propose that FAPs should be
purified or under red dim light conditions.
(Fig. 6B). Due to the relatively low solubility of palmitic acid in buffer,
ethanol was used as a co-solvent to dissolve substrate for steady-state
assays. Over the substrate range (0–500 μM palmitic acid) used, Mi-
chaelis-Menten behaviour is observed. Fitting to the standard Mi-
chaelis-Menten equation produced an apparent K
M
value for palmitic
acid of 98.8 ± 53.3 μM. The apparent kcat determined using gas
−
1
chromatography was 0.31 ± 0.06 s ; the catalytic efficiency (kcat
/
K
M
) is therefore 3.1 ± 2.7 s mM−1. These data now provide a
−
1
platform for additional steady-state characterisation of CvFAP (e.g.
using a range of substrates of differing chain lengths and/or deutera-
tion) to gain further insights into kinetic mechanism and substrate
specificity of CvFAP and related photodecarboxylases.
To investigate further the mechanism of photoinactivation, we
performed EPR spectroscopy of CvFAP that had been inactivated by
blue light irradiation in absence of FA substrate. The CW-EPR spectrum
of inactivated CvFAP compared to a CvFAP sample that was kept in the
dark are strikingly different. No organic radical signal was recorded in
the ‘dark’ sample. However, after 30 min blue light exposure (455 nm
LED), a large radical signal is observed in the EPR spectrum (Fig. 5).
This signal will likely have contributions from both the FAD semi-
quinone and a protein-based organic radical(s) (e.g. amino acid radicals
such as glycine, cysteine, tyrosine, or tryptophan). The chemical nature
of these species remains to be determined. That said, the EPR spectra
obtained indicate that radical accumulation correlates with photo-
inactivation, and is likely caused by electron transfer from active site
residues to photoexcited FAD singlet state following blue light illumi-
nation. Glutathione (GSH) – an antioxidant that protects free radical
damage to the cell [12] – did not protect CvFAP from blue light-induced
inactivation. Addition of 1 mM or 2 mM GSH before and after illumi-
nation for 30 min had no discernible effect on CvFAP inactivation.
Protein based radicals are known to modify protein activity by
cross-linking, adduct formation and backbone cleavage [13,14]. The
formation of protein-based radicals would represent an ‘off pathway’
reaction that occurs when FA is not present in the active site (Fig. 5B).
In the Michaelis complex, the presence of substrate provides a com-
peting ‘on pathway’ route for electron donor to the photoexcited flavin
originating from the FA carboxyl group. Ensuring the active site is al-
ways saturated with FA substrate should help to protect CvFAP from
photoinactivation. In the cell, therefore, the abundance of cytosolic FAs
could help to suppress inactivation [15].
4. Conclusion
As a natural light-activated enzyme that can decarboxylate fatty
acids, FAP has the potential to play an important biotechnological role
in the chemical and energy sectors. Here, we have described methods
for the production of active, pure samples of FAP. Enzyme purification
needs to be conducted in the dark to minimise enzyme inactivation by
light. Without substrate (e.g. palmitic acid), excitation of the flavin
leads to formation of protein-based organic radicals and enzyme in-
activation. These radical species are linked to local changes around the
flavin cofactor and not overall secondary structural changes. Binding of
substrate (palmitic acid) prevents enzyme inactivation. Substrate
binding (lauric acid) also leads to oligomerisation to form a hexameric
form of CvFAP. The described FAP sample preparations have, for the
first time, enabled determination of apparent steady-state kinetic con-
stants for palmitic acid substrate, which is the presumed natural sub-
strate of the enzyme. These new preparative methods for enzyme iso-
lation should now support detailed biophysical investigations of kinetic
and chemical mechanisms of FAPs and their exploitation in bio-
technology.
CRediT authorship contribution statement
Balaji Lakavath: Investigation. Tobias M. Hedison: Investigation,
Conceptualization, Writing - original draft, Writing - review & editing,
Supervision. Derren J. Heyes: Investigation, Writing - review &
editing, Supervision, Conceptualization. Muralidharan Shanmugam:
Investigation. Michiyo Sakuma: Investigation. Robin Hoeven:
Investigation. Viranga Tilakaratna: Investigation. Nigel S. Scrutton:
Writing - original draft, Writing - review & editing, Supervision,
Funding acquisition, Conceptualization.
To prevent inactivation, efforts should be made to ensure the active
site of CvFAP is saturated with substrate. This is relevant to exploitation
of FAPs in microbial cell factory engineering and also cell-free bioca-
talytic applications. For example, protein-engineering applications
would need to ensure tight binding of FAs to ensure enzyme saturation
to suppress inactivation. Similarly, continuous feeding of FAs to FAPs
might also suppress photoinactivation.
3.3. Kinetic parameters of CvFAP-catalysed decarboxylation
Acknowledgements
Previous biocatalytic studies have shown that palmitic acid (C16) is
This work was supported by the Future Biomanufacturing Research
Hub (grant EP/S01778X/1), funded by the Engineering and Physical
Sciences Research Council (EPSRC) and Biotechnology and Biological
Sciences Research Council (BBSRC) as part of UK Research and
Innovation. Balaji Lakavath thanks the Indian Ministry of Higher
Education for funding. The BBSRC grant BB/N013980/1helped to
support this project.
the preferred substrate for CvFAP [1]. To date, however, studies have
been performed with CvFAP in broken and/or clarified cell lysates.
There has been no analysis of the steady-state kinetic parameters for
decarboxylation with active, purified enzyme. Here, we used gas
chromatography to determine steady-state kinetic parameters asso-
ciated with CvFAP-catalysed decarboxylation of palmitic acid.
We demonstrated above that blue light irreversibly inactivates FAP
in absence of FA substrate. It is of interest to also explore if blue light
exposure inactivates CvFAP when bound to FA substrate. Exploring how
blue light intensity influences the steady-state turnover is one means of
assessing this aspect of CvFAP behaviour. Fig. 6A shows a linear re-
lationship between blue light intensity and CvFAP-catalysed dec-
Appendix A. Supplementary data
−
2
−1
arboxylation over a range of light intensity (0–2000 μmol m
s
).
References
This linear dependence is consistent with substrate binding inhibiting
light-dependent inactivation and with electron transfer predominantly
taking place along the catalytic ‘on pathway’.
Next, we investigated steady-state kinetic behaviour of CvFAP at a
fixed light intensity over a range of palmitic acid concentrations
5