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N Enzymes
N Enzymes
Evidence
That S-Adenosylmethionine Binds in Contact with
the
Catalytically Active [4Fe-4S]+ Cluster of Pyruvate
Formate-Lyase Activating Enzyme
Introduction
●
Materials
●
● Methods
Data
●
Conclusion
●
Introduction
● Pyruvate formate-lyase
activating enzyme (PFL-AE)
utilizes iron-sulfur clusters and
S-adenosylmethionine (AdoMet)
to initiate radical catalysis
● Stable glycyl radical at G734 of
PFL
● Reductive cleavage of AdoMet
● Yields methionine and 5'-
deoxyadenosine (5'-dAdo)
● PFL converts pyruvate and CoA
to formate and acetyl-CoA
Introduction
● Purified PFL-AE contains mostly cuboidal
[3Fe-4S]+ which are converted to [4Fe-4S]
2+/+ clusters in reducing conditions.
● The [4Fe-4S]+ form is the essential and
active form
● Carried out EPR and ENDOR studies of
the PFL-AE -AdoMet complex to monitor
the interaction of AdoMet with the [4Fe-
4S] cluster
Materials and Methods
● PFL-AE was purified from E. coli
● AdoMet was labeled with 2H (CD3-AdoMet)
● 87.9% yield
● And with 13 C (13 CH3-AdoMet)
● 86.0% yield
● Prepared [4Fe-4S]+PFL-AE for EPR and
[4Fe-4S]2+PFL-AE for ENDOR analysis
EPR Results
● A: PFL-AE [4Fe-
4S]1+ without
AdoMet
● B: PFL-AE [4Fe-
4S]1+with bound
AdoMet
2
H ENDOR Data
(a) and (d)
photoreduced
sample;
(b) cryoreduced
sample
(c) and (e)
Simulations (dashed
lines) with dipolar A
tensors
13
C ENDOR DATA