Supplementary Table I. Fold Change in Kinetic Constants Relative to WT pfMDH on DMannitolǂ
Decrease in kcat Increase in KM Decrease in kcat/KM
Enzyme
(s-1)
(mM)
(M-1s-1)
E133A
1.1
3.1
3.4
E133Q
1.4
2.5
3.4
N191A
210
0.8 (Decrease)
160
D230A
11
7.2
81
K295A
ND
ND
ND
N300A
21
12
240
N300D
5300
3.4
18000
N300S
46
8.4
380
H303A
9.0
16
140
R373A
240
3.0
720
K381A
23
4.0
95
H303A+R373A+K381A
4400
1.4
4600
ǂKinetic
data calculated from eight data points obtained in triplicate by measuring the
accumulation of NADH at a wavelength of 340nm during the oxidation of D-mannitol over
the period of an hour. Assay was performed in 100µL 50mM HEPES with 150mM NaCl
(pH 7.4), 1mg/ml BSA, 1mM NAD+, and 90-900nM pfMDH. Two-fold serial dilutions were
performed on the substrate where the maximum final concentration of D-mannitol was
200mM. ND = No detection, detection limit of 0.001 M-1s-1.
Supplementary Table II. Fold Change in Kinetic Constants Relative to WT pfMDH on DArabitolǂ
Decrease in kcat Increase in KM Decrease in kcat/KM
Enzyme
(s-1)
(mM)
(M-1s-1)
E133A
0.9 (Increase)
2.4
2.2
E133Q
1.2
2.3
2.7
N191A
150
2.3
340
D230A
2300
0.4 (Decrease)
840
K295A
ND
ND
ND
N300A
72
2.4
170
N300D
5100
1.7
8400
N300S
110
2.3
250
H303A
23
2.6
59
R373A
190
2.6
490
K381A
18
3.2
57
H303A+R373A+K381A
3900
0.6 (Decrease)
2300
ǂKinetic data calculated from eight data points obtained in triplicate by measuring the
accumulation of NADH at a wavelength of 340nm during the oxidation of D-arabitol over
the period of an hour. Assay was performed in 100µL 50mM HEPES with 150mM NaCl
(pH 7.4), 1mg/ml BSA, 1mM NAD+, and 90-900nM pfMDH. Two-fold serial dilutions were
performed on the substrate where the maximum final concentration of D-arabitol was
200mM. ND = No detection, detection limit of 0.001 M-1s-1.
Supplementary Table III. Fold Change in Kinetic Constants Relative to WT pfMDH on
Meso-Erythritolǂ
Enzyme
Decrease in kcat/KM (M-1s-1)
E133A
1.1
E133Q
1.2
N191A
510
D230A
ND
K295A
ND
N300A
170
N300D
ND
N300S
250
H303A
37
R373A
ND
K381A
ND
H303A+R373A+K381A
ND
ǂKinetic data calculated from eight data points obtained in triplicate by measuring the
accumulation of NADH at a wavelength of 340nm during the oxidation of meso-erythritol
over the period of an hour. Assay was performed in 100µL 50mM HEPES with 150mM
NaCl (pH 7.4), 1mg/ml BSA, 1mM NAD+, and 90-900nM pfMDH. Two-fold serial
dilutions were performed on the substrate where the maximum final concentration of Darabitol was 200mM. ND = No detection, detection limit of 0.001 M-1s-1. Since saturation
was not observed for both WT and mutant pfMDH on meso-erythritol, only the fold
change in kcat/KM is reported.
Supplementary Table IV. Unit Concentrations of pfMDH in Kinetic Assays
Enzyme
Units (µmol NADH released/min)
WT
2.26 * 10-3
E133A
1.78 * 10-3
E133Q
1.52 * 10-3
N191A
3.03 * 10-4
D230A
1.73 * 10-3
H303A
1.28 * 10-3
N300A
3.79 * 10-4
N300D
1.64 * 10-5
N300S
1.20 * 10-3
R373A
5.83 * 10-5
K381A
3.98 * 10-4
H303A, R373A, K381A
5.26 * 10-6
Units of WT and mutant pfMDH used in the kinetic assays were calculated from the
steady-state oxidation of D-mannitol at an initial concentration of 100mM. The
accumulation of NADH was measured at a wavelength of 340nm in 100µL 50mM HEPES
containing 150mM NaCl (pH 7.4), 1mg/ml BSA, and 1mM NAD+.
Supplementary Figure S1. DNA sequence (A) and corresponding amino acid sequence (B)
used to clone and express WT pfMDH.
A.
ATGAAACTGAACAAACAGAATCTGACCCAGCTGGCACCGGAAGTTAAACTGCCTG
CATATACCCTGGCAGATACCCGTCAGGGTATTGCACATATTGGTGTTGGTGGTTTTC
ATCGTGCACATCAGGCATATTATACCGATGCACTGATGAATACAGGTGAAGGTCTGG
ATTGGAGCATTTGCGGTGTTGGTCTGCGTAGCGAAGATCGTAAAGCACGTGATGATC
TGGCAGGTCAGGATTACCTGTTTACCCTGTATGAACTGGGCGATACCGATGATACCG
AAGTTCGTGTTATTGGTAGCATTAGCGATATGCTGCTGGCAGAAGATAGCGCACAGG
CACTGATTGATAAACTGGCAAGTCCGGAAATTCGTATTGTTAGCCTGACCATTACCG
AAGGTGGTTATTGTATTGATGATAGCAACGGTGAATTTATGGCACATCTGCCGCAGA
TTCAGCACGATCTGGCACATCCGAGCAGCCCGAAAACCGTTTTTGGTTTTATTTGTG
CAGCACTGACACAGCGTCGTGCAGCAGGTATTCCGGCATTTACCGTTATGAGCTGTG
ATAACCTGCCGCATAATGGTGCAGTTACCCGTAAAGCCCTGCTGGCATTTGCAGCCC
TGCATAATGCAGAACTGCATGATTGGATTAAAGCCCATGTTAGCTTTCCGAATGCAA
TGGTTGATCGTATTACCCCGATGACCAGCACCGCACATCGTCTGCAACTGCATGACG
AACATGGTATTGATGATGCATGGCCTGTTGTTTGTGAACCGTTTGTTCAGTGGGTTCT
GGAAGATAAATTTGTTAATGGTCGTCCGGCATGGGAAAAAGTTGGTGTTCAGTTTAC
CGATGATGTTACCCCGTATGAAGAAATGAAAATTGGTCTGCTGAATGGTAGCCATCT
GGCACTGACCTATCTGGGTTTTCTGAAAGGTTATCGTTTTGTGCATGAAACCATGAA
CGATCCGCTGTTTGTTGCCTATATGCGTGCATATATGGATCTGGATGTGACCCCGAA
TCTGGCTCCGGTTCCGGGTATTGATCTGACCGATTATAAACAGACCCTGGTTGATCG
TTTTAGCAATCAGGCAATTGCAGATCAGCTGGAACGTGTTTGTAGTGATGGTAGCAG
CAAATTTCCTAAATTTACCGTTCCGACAATTAATCGTCTGATTGCAGATGGTCGTGA
AACCGAACGTGCAGCACTGGTTGTTGCAGCATGGGCACTGTATCTGAAAGGCGTTG
ATGAAAATGGTGTGAGCTATACCATTCCTGATCCGCGTGCAGAATTTTGTCAGGGTC
TGGTTTCAGATGATGCACTGATTAGCCAGCGTCTGCTGGCAGTTGAAGAAATTTTTG
GCACCGCAATTCCGAATAGTCCGGAATTTGTTGCCGCATTTGAACGTTGTTATGGTA
GCCTGCGTGATAATGGTGTTACCACCACCCTGAAACATCTGCTGAAAAAACCTGTTG
GTAGCCTCGAGCACCACCACCACCACCACTGA
B.
MKLNKQNLTQLAPEVKLPAYTLADTRQGIAHIGVGGFHRAHQAYYTDALMNTGEGL
DWSICGVGLRSEDRKARDDLAGQDYLFTLYELGDTDDTEVRVIGSISDMLLAEDSAQAL
IDKLASPEIRIVSLTITEGGYCIDDSNGEFMAHLPQIQHDLAHPSSPKTVFGFICAALTQRR
AAGIPAFTVMSCDNLPHNGAVTRKALLAFAALHNAELHDWIKAHVSFPNAMVDRITPM
TSTAHRLQLHDEHGIDDAWPVVCEPFVQWVLEDKFVNGRPAWEKVGVQFTDDVTPYE
EMKIGLLNGSHLALTYLGFLKGYRFVHETMNDPLFVAYMRAYMDLDVTPNLAPVPGID
LTDYKQTLVDRFSNQAIADQLERVCSDGSSKFPKFTVPTINRLIADGRETERAALVVAA
WALYLKGVDENGVSYTIPDPRAEFCQGLVSDDALISQRLLAVEEIFGTAIPNSPEFVAAF
ERCYGSLRDNGVTTTLKHLLKKPVGSLEHHHHHH
Supplementary Table S1. Ascension numbers of 184 non-redundant sequences identified to be
between 36% and 80% homologous to WT pfMDH.
515702451
515535807
518239409
495642902
146309486
498497260
494275963
146282546
15597538
498173937
522174752
521075588
226944812
589830556
522170660
494260563
571266873
434394911
518339205
428209636
516898902
493683979
575466942
92112776
262197719
407643942
284029330
515856642
94972329
494171420
556568052
336119172
546177344
512441446
586941878
551296630
494601864
517849719
518415081
495633931
498315174
515333324
495986283
518796391
494896875
498278369
493639306
497463055
518858980
271964979
340794648
152964270
516016174
516488122
551261793
490119964
307545080
495967966
497825909
495608014
258654087
491343912
378719990
111019801
490717977
488722889
545328676
226357932
518988023
537741123
109897106
333922052
220912479
496384922
308175969
489897043
496119188
495952717
516626505
490946336
517840686
494001700
586971629
284991238
77362123
488731242
496491451
375140369
515039701
565827178
578035162
492507053
498801695
522004273
515077277
343084236
32471932
517463061
496109292
493369601
518857916
488708700
518001887
515744812
497569714
493877150
517145004
551305719
551303028
162146708
522057298
383315884
310819331
495304587
550958012
241206434
493994630
497926326
226307177
498348458
340620224
518840934
522081866
347761280
319954108
527074656
114563074
357386909
590080478
152968148
470157564
451942875
516499917
493595526
551288914
518949951
516725859
551281193
108801323
588489233
333892785
489594618
516539804
544665018
516861632
490023900
392418692
517455113
497450240
557723517
497956595
493503524
498211373
552765122
336173634
495157575
518289561
212534026
325287783
429855165
550807130
497434561
493246423
496141395
489084599
494944720
543892134
320589110
516823997
296395256
495524010
584371390
367015078
496377603
590027327
496427861
510825090
498235202
496142751
584423808
389645703
425765994
497433346
Supplementary Figure S2. Michaelis-Menten regression plots of experimentally observed
velocity versus D-mannitol concentration used to generate Michaelis-Menten constants for WT
pfMDH and mutants.
9
10
11
Supplementary Figure S3. Michaelis-Menten regression plots of experimentally measured
velocity versus D-arabitol concentration used to generate Michaelis-Menten constants for WT
pfMDH and mutants.
12
13
14
Supplementary Figure S4. Linear regression plots of experimentally measured velocity versus
meso-erythritol concentration used to generate Michaelis-Menten constants for WT pfMDH and
mutants.
15
16
Supplementary Figure S5. First order Arrhenius equations and constants used to calculate
changes in (A) kcat, (B) KM, and (C) kcat/KM for pfMDH mutants on D-mannitol, D-arabitol, and
meso-erythritol. (D) Equation used to determine the change in free energy between WT and mutant
pfMDH. All free energy values were calculated in joules and converted into kcal/mole.
A.
𝑘ℎ
𝛥𝐺 = −𝑅𝑇𝑙𝑛 (
)
k𝐵 𝑇
R = 8.314 J mol-1 K-1
T = 298 K
h = 6.626 x 10-34 J s
kB = 1.38 x 10-23 J K-1
k = kcat (s-1)
B.
𝛥𝐺 = −𝑅𝑇𝑙𝑛(K M )
R = 8.314 J mol-1 K-1
T = 298 K
C.
𝑘ℎ𝑐 °
𝛥𝐺 = −𝑅𝑇𝑙𝑛 (
)
k𝐵 𝑇
R = 8.314 J mol-1 K-1
T = 298 K
h = 6.626 x 10-34 J s
kB = 1.38 x 10-23 J K-1
c° = 1 M
17
k = kcat/KM (M-1s-1)
D.
𝛥𝛥𝐺𝑘𝑐𝑎𝑡 = 𝛥𝐺𝑀𝑢𝑡𝑎𝑛𝑡 𝑘
𝑐𝑎𝑡
− 𝛥𝐺𝑊𝑇 𝑘
𝑐𝑎𝑡
𝛥𝛥𝐺𝐾𝑀 = 𝛥𝐺𝑀𝑢𝑡𝑎𝑛𝑡 𝐾 − 𝛥𝐺𝑊𝑇 𝐾
𝑀
𝛥𝛥𝐺𝑘𝑐𝑎𝑡⁄
𝐾𝑀
= 𝛥𝐺𝑀𝑢𝑡𝑎𝑛𝑡 𝑘𝑐𝑎𝑡⁄
𝐾𝑀
𝑀
− 𝛥𝐺𝑊𝑇 𝑘𝑐𝑎𝑡⁄
𝐾𝑀
18