Michael K. Jones characterized the enzymes involved in methanofuran biosynthesis in the archaeon Methanocaldococcus jannaschii. The MJ1099 gene encodes an aldolase domain protein called MfnB that was shown to condense two molecules of glyceraldehyde-3-phosphate into 4-(hydroxymethyl)-2-furancarboxaldehyde-phosphate, a precursor of methanofuran. MfnB was cloned, expressed, purified, and its activity was tested and optimized. Site-directed mutagenesis identified amino acids important for MfnB activity. The crystal structure of MfnB was determined, revealing an α/β-barrel fold typical of aldolase enzymes.

1. Michael K. Jones
Date: July 31, 2014
Advised by: Dr. Robert H. White
Email: mikej10@vt.edu
Biosynthesis of
Methanofuran in
Methanocaldococcus
jannaschii

2. • Thermophilic archaea produce 350 million tons of methane a year
• Uses methanogenic coenzymes to reduce CO2 to methane
Methanocaldococcus jannaschii
http://wishart.biology.ualberta.ca/BacMap/cg
i/getSpeciesCard.cgi?accession=NC_000909&r
ef=index_12.html
Goal: Characterize the enzymes
involved in coenzyme biosynthesis,
especially methanofuran biosynthesis

3. Methanogenesis
“Biochemistry- The Chemical Reaction of Living Cells”, Vol. 1 P814 H4-Methanopterin
F 420
F 430
Coenzyme M
Methanofuran
Methanogenic coenzymes
7-Mercaptoheptanoylthreonine
phosphate

4. Methanofuran Biosynthesis
• MJ1099 gene product
(MfnB) is known to
contain a class I
aldolase domain
• MfnB was proposed
to condense
glyceraldehyde-3-
phosphate (GA-3P) to
form 4-
(hydroxymethyl)-2-
furancarboxaldehyde-
phosphate (4-HFC-P)
MJ1099
(MfnB)

5. • E. coli transformed with MJ1099 and
expressed
• Protein extracted from cells and purified
with ion exchange column
Cloning, expression and purification of
MJ1099 in BL 21 E. coli cells
http://www.addgene.org/plasmid_protocols/bacterial_transformation/

6. • Expressed and purified
protein fractions tested
for protein concentration
with SDS gel
electrophoresis
• Enzyme identity verified
by MALDI mass spec.
analysis of tryptic
peptides of the excised
protein band
Protein
standard 0 250 340 370 410 470 500 530 790
97.0 kDa
66.2 kDa
45.0 kDa
31.0 kDa
21.5 kDa
14.9 kDa
Elution Concentration of NaCl (mM)
SDS Gel of MJ1099 product (MfnB)

7. Bradford Protein Assay
0
0.068
0.191
0.243
0.354
0.406y = 0.0464x
R² = 0.9409
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0 2 4 6 8 10 12
Absorbanceat595nm(A)
Protein Concentration (ng/µL)
Elution
Concentration of
NaCl (mM) A595 ng/µl in the tube
397 0.0106 45.6896552
410 0.5938 2559.48276
431 1.0818 4662.93103
448 0.6232 2686.2069
470 0.6694 2885.34483
483 0.4275 1842.67241
500 0.1644 708.62069
517 0.0343 147.844828
530 0 0
551 0.0427 184.051724
• Protein concentration was determined from
protein standard curve created with known
concentrations of protein (BSA)

8. • MfnB was shown to react with 2
molecules of glyceraldehyde-3-
phosphate (GA-3P) to form 4-
(hydroxymethyl)-2-
furancarboxaldehyde-phosphate
(4-HFC-P)
MfnB Substrate Specificity
Figure 6. (A) HPLC analysis of synthetic 4-HFC. (B)
Enzymatic reaction product generated by MfnB. (C)
Resulting product from the MfnB reaction treated
with phosphatase.
Miller, D.; Wang, Y.; Xu, H.; Harich, K.; White, R. H., Biosynthesis of the
5-(Aminomethyl)-3-furanmethanol Moiety of Methanofuran.
Biochemistry 2014, 53 (28), 4635-4647.

9. • MfnB product formation can be measured at 280 nm, estimating
molar absorptivity of 4-HFC-P with a 5-HFC calibration curve
ε = 15933 M-1 cm-1
MfnB Product 4-HFC-P
y = 15.933x
R² = 0.9966
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 0.02 0.04 0.06 0.08 0.1 0.12Absorbanceat280nm
5-HFC Concentration (mM)

10. pH-Dependent study of MfnB
• pH curve determined by calculating 4-
HFC-P formation at varying pH from 4.0
to 11.0.
• Highest relative activity in 50 mM MES
buffer pH = 7.0
• MfnB is metal-independent.

11. Kinetic study of MfnB
• V0 = Vmax [S] / KM + [S]
• kcat = 0.023 ± 0.002 s-1
• KM = 0.05 ± 0.02 mM
K155R

12. Site-directed Mutagenesis
• Variants D25N, K27R, K85R
and D151N showed
complete inactivity
• K155R retained ~80%
activity
K155R
M. jannaschii
M. fervens
M. okinawensis
M. smithii
M. sp.
M. kandleri
M. mazei
S. roseosporus
M. versatilis
M. extorquens
M. sp.
M. jannaschii
M. fervens
M. okinawensis
M. smithii
M. sp.
M. kandleri
M. mazei
S. roseosporus
M. versatilis
M. extorquens
M. sp.
M. jannaschii
M. fervens
M. okinawensis
M. smithii
M. sp.
M. kandleri
M. mazei
S. roseosporus
M. versatilis
M. extorquens
M. sp.
D25 K27
K85
K155D151

13. MfnB Crystal Structure
• MfnB exhibits a typical TIM (α/β)8 barrel fold of the aldolase superfamily

14. • Proposed mechanism for
aldol condensation of
2(GA-3P) to 1(4-HFC-P)
O
OP
O
H
HO
OP
O
DHAP
15N-alanine
O
OP
H3
15N
F1-P
4-HFC-P
MJ0684
H2O
AAT
lysine-27-NH2
Pi
B:
O O
OPO
HO
OP
H
N
OH
O
lysine-27
1 July 14
H2O
O
OPHO
H NH2-lysine-85 strong base
O
OP
O
AH
H H :B
HO
OPHO
H
B:
HA
H
AH
:B
1. Other conserved lysines
and arginine bind the
phosphates
2. I say the enyme has
two binding sites for G-3-P
alanine
pyruvate
H2O
glyceraldehyde-3-P glyceraldehyde-3-P
enediol
Reaction site I
Reaction site II
O
OPO
Inhibitor of site II. When
bound blocks condensation
reaction and allows site I to
produce MG and lable lysine-
198
O
OPHO
H
HO
O
O
O
O
lysine-27
H
N
+
O
lysine-27
H
N
two possible pathways to enediol
+
O
D22
D25
I see one binding site for Ga-3P in the active
site that produces the enediol. The first molecule
to bind looses Pi and forms methylglyoxal that
binds to lysine-27 as a Schiff base. This
molecule then moves to a second part of the active
site and another Ga-P binds and reacts like the first
to forms a second enediol that rearranges to DHAP.
The MG and DHAP undergoes an aldol condensation
that after the lose of two waters produces 4-HFC-P.
The lose of the 2d water requires a strong base that is
supplied by K85.
H
B:
enediol
O
OP
O
AH
H H :B
D22
D25
MGo

15. • Fralin Life Science Institute; Summer Undergraduate Research
Fellowship
• Dr. Robert H. White
• Dr. Yu Wang
• Dr. Kylie. Allen
• Danielle Miller
• Huimin Xu
Acknowledgements