Supplemental Materials Table S1. Operators used in reaction network generation Operator
Operator action OH
1.1.1.a
NAD +
+
W1
W2
O + NADH
H
W1
+
H+
W2
W 1 and W2 can only be carbon or hydrogen. 1.1.-1.a
Exact reverse of 1.1.1.a OH
1.1.1.b
W
W
W2 C
C W2
O All W's can be anything. 1.1.-1.b
Exact reverse of 1.1.1.b OH
1.1.1.c
O
OH W W WW W W
CW3 or H
C W2
W
O
W2 C
C W2
CW3 or H
W W W W
O
W W
OH W W W
All W's can be anything. 1.1.-1.c
1.1.1.d
Exact reverse of 1.1.1.c
W2
O OHO C OH
+ NADH +
OH O W1 OH C NAD+ W2 OH+ H
H+
W1
W1 must be an ethyl or methyl group. W2 can only be carbon. O
1.2.-1.b
W1
+ CoA
NADH +
O
H+ W1
+ CoA H
+
NAD+
W1 can be carbon or long carbon chain, NAD+ and NADH can be NADP+ and NADPH. W1
1.2.7.a
O
W1 + CoA + 2 ferredoxin[ox]
O
OH
CoA + CO2 + 2 H+ + 2 ferredoxin[red] O
W 1 can be carbon, oxygen, sulfer and hydrogen.
1.3.1.a
1.3.-1.a
O or CW 2 or NW H H + NAD + W W W W All W's can be any type of atom.
NADH
+
O or CW2 or NW W + H+ W W W
Exact reverse of 1.3.1.a
1|Page
O
W
W W
HO 1.3.1.b
NAD +
W
+
W
W
+
NADH +
W
W
H
W
W
H + + CO 2
W W
All W's can be any type of atom. OH
OH
HO 1.3.1.c
W
HO
W
NAD + +
+ W
W W All W's can be anything.
NADH + 1.3.1.d
W1
W2
W1
W2
+
+ H
W
H+
NADH +
W W
+
W1 H H
NAD +
W1
W2 W2
W1 must be carbon. W2 can be anything. 1.3.-1.d
1.3.1.e
1.3.-1.e
Exact reverse of 1.3.1.d W
W
W +
W W W must be OH or halogen.
W
NADH
+
H+
W
Exact reverse of 1.3.1.e W1
1.3.1.g
W +
NAD +
H
W1 H H
+ NADH + H + H
W2
H H W2
+ NAD+
W 1 can only be carbon, W2 can be anything. O 2.3.1.c
CoA
+
OH
W1
O
OH +
W2
W1
W1
W1
CoA
W2
W1 can be C or H and W2 can be S and H. O or CW2 or NW
2.3.1.f
CoA
W
O or CW2 or NW
+ CoA
CH3
CoA
O or CW2 or NW
H 2C
O or CW 2 or NW
+ CoA
W W can be hydrogen or carbon-based substitent of any length.
2|Page
2.3.1.g
O
O
O +
O
+ CoA C W H2 W can be hydrogen or carbon-based substitent of any length. CoA
CoA
W
CoA
CH 3
OH O 2.3.3.a
W
O +
W
CoA
+
W
H 2O
Exact reverse of 2.3.3.a O
4.2.1.a
+ CoA
O
W can only be either carbon or hydrogen. 2.3.-3.a
W
HO
W1
O
W H
W
W
O +
W OH
W
W
W
W W
W 1 can only be C, S, H, and O. All other W's unrestricted. 4.2.-1.a
Exact reverse of 4.2.1.a O
4.2.1.b
H
O
W CoA W W OH
H 2O +
CoA
W W
W
W can be anything. 4.2.-1.b
Exact reverse of 4.2.1.b O W W 2 2
4.2.1.c
+ W1 OH
HO H NH2
W O W + HO W H NH2 O
OH
OH
W1-OH is limited to water. W2 can be anything.
4.2.-1.c
4.2.1.d
Exact reverse of 4.2.1.c W1
O
O
H
W2 + W2 NH2
HO
H N
O W2 HO H
NH + HO W1
W2 NH2
-OW1 leaving group can also be SW1. W2 can be anything.
4.2.-1.d
Exact reverse of 4.2.1.d
W1
4.2.1.e
OH H H
OH
W2
H2O +
W1
O W2
W1 can only be H or OH. W2 can only be H or CH3.
3|Page
O
4.2.1.f
H2O + W
N W
NH2
W can be anything.
W
W
W
W
W
W
W
W
+ 4.2.-1.g
H2 O
W can be carbon or hydrogen. O
4.2.-1.h
W2
W1
H2 O
+
W1
W2
H
W 1 can be any atom. W 2 is C or N.
4.2.2.a
H2O
+
OW
OH C W2
O C CW 3 W2
OH CW 3
+
WO
W can be anything including H. O W O P O 2
W2
4.2.3.a
H2 N W1 H
O HO P O W2 + O W2
O W2 W2
H2N
W2
W1
W2
W can be anything. O W O P O W O W H W W
4.2.3.b
W O
O HO P O W + O W
O
W
W
W W
W can be anything.
4.2.-3.b
Exact reverse of 4.2.3.b O W O P O W H O W
4.2.3.c
W
W
W W W
W O W W HO P O W+ W O W W W
W can be anything.
4.2.-3.c
Exact reverse of 4.2.3.c
4|Page
Table S2. KEGG reactions that can be reproduced by the selected operators R00205 R00264
R01293 R01294
R01735 R01738
R02347 R02349
R02756 R02762
R03114 R03119
R03544 R03545
R04065 R04088
R04552 R04556
R04862 R04880
R05240 R05241
R00300
R01333
R01739
R02352
R02776
R03123
R03572
R04099
R04557
R04882
R05243
R00342
R01337
R01740
R02353
R02777
R03155
R03577
R04105
R04681
R04883
R05271
R00343
R01338
R01745
R02377
R02792
R03169
R03581
R04106
R04682
R04888
R05272
R00351
R01361
R01747
R02395
R02793
R03176
R03582
R04115
R04697
R04889
R05275
R00352
R01370
R01752
R02401
R02799
R03177
R03583
R04136
R04724
R04891
R05282
R00402
R01371
R01763
R02413
R02800
R03191
R03584
R04139
R04725
R04892
R05283
R00465
R01388
R01773
R02462
R02819
R03192
R03585
R04151
R04737
R04903
R05286
R00472
R01392
R01775
R02463
R02820
R03198
R03586
R04185
R04739
R04910
R05289
R00604
R01403
R01781
R02471
R02832
R03226
R03591
R04186
R04741
R04916
R05305
R00703
R01404
R01813
R02472
R02834
R03227
R03592
R04198
R04743
R04917
R05310
R00704
R01414
R01825
R02476
R02836
R03234
R03636
R04199
R04745
R04953
R05311
R00710
R01415
R01836
R02477
R02841
R03251
R03667
R04203
R04748
R04954
R05312
R00711
R01419
R01838
R02527
R02855
R03261
R03707
R04210
R04753
R04955
R05313
R00713
R01420
R01866
R02528
R02856
R03264
R03709
R04236
R04757
R04956
R05315
R00714
R01429
R01869
R02531
R02878
R03278
R03713
R04278
R04758
R04958
R05347
R00717
R01430
R01899
R02536
R02892
R03279
R03716
R04279
R04778
R04959
R05348
R00732
R01446
R01921
R02537
R02893
R03290
R03733
R04285
R04805
R04961
R05349
R00733
R01451
R01932
R02544
R02940
R03292
R03745
R04304
R04810
R04962
R05350
R00746
R01456
R01971
R02545
R02941
R03300
R03753
R04307
R04812
R04964
R05353
R00754
R01457
R01975
R02548
R02944
R03302
R03759
R04309
R04817
R04965
R05354
R00812
R01465
R01976
R02553
R02945
R03324
R03763
R04310
R04818
R04966
R05395
R00814
R01486
R01977
R02565
R02946
R03325
R03764
R04330
R04819
R04967
R05396
R00835
R01487
R01978
R02566
R02954
R03336
R03776
R04344
R04823
R04969
R05397
R00842
R01488
R01986
R02577
R02969
R03337
R03790
R04360
R04824
R04970
R05575
R00844
R01505
R02032
R02580
R02975
R03338
R03792
R04370
R04829
R04996
R05576
R00845
R01513
R02034
R02593
R02978
R03339
R03827
R04375
R04830
R05038
R05581
R00871
R01520
R02039
R02609
R02988
R03357
R03845
R04387
R04831
R05049
R05582
R00904
R01521
R02047
R02610
R02989
R03373
R03847
R04412
R04832
R05050
R05639
R00919
R01524
R02123
R02611
R02993
R03396
R03848
R04418
R04833
R05066
R05641
R00977
R01525
R02124
R02637
R02994
R03397
R03856
R04419
R04834
R05068
R05643
R00978
R01542
R02177
R02639
R03012
R03398
R03869
R04426
R04835
R05099
R05649
R00998
R01543
R02207
R02640
R03021
R03399
R03886
R04429
R04836
R05119
R05663
R01000
R01550
R02208
R02651
R03022
R03401
R03889
R04430
R04837
R05124
R05664
R01034
R01604
R02209
R02655
R03043
R03406
R03900
R04440
R04838
R05150
R05678
R01036
R01607
R02229
R02678
R03044
R03407
R03918
R04453
R04839
R05231
R05679
R01039
R01608
R02252
R02679
R03048
R03431
R03930
R04506
R04840
R05232
R05680
R01041
R01644
R02257
R02680
R03051
R03495
R03942
R04517
R04842
R05233
R05681
R01058
R01689
R02258
R02683
R03054
R03497
R03945
R04518
R04843
R05234
R05682
R01096
R01704
R02259
R02684
R03080
R03498
R03962
R04533
R04844
R05235
R05683
5|Page
R01163
R01708
R02260
R02695
R03102
R03506
R03963
R04536
R04845
R05237
R05684
R01183
R01728
R02343
R02697
R03103
R03507
R03989
R04543
R04846
R05238
R05685
R01213
R01730
R02344
R02736
R03104
R03520
R04049
R04544
R04847
R05239
R05686
R05687
R06117
R06785
R06983
R07417
R07804
R08105
R08544
R01202
R03317
R07150
R05689
R06126
R06832
R06985
R07418
R07890
R08140
R08557
R01694
R03826
R07728
R05690
R06166
R06846
R07015
R07428
R07894
R08141
R08558
R01758
R05069
R08310
R05691
R06180
R06847
R07060
R07437
R07898
R08194
R08573
R01895
R05071
R02906
R05692
R06272
R06857
R07104
R07441
R07936
R08198
R08590
R01896
R05147
R03322
R05693
R06286
R06891
R07136
R07442
R07952
R08226
R00286
R01903
R05571
R03463
R05694
R06366
R06896
R07137
R07483
R07985
R08281
R00421
R01904
R05604
R05308
R05699
R06407
R06910
R07138
R07495
R07998
R08282
R00501
R02441
R05607
R06927
R05700
R06408
R06914
R07139
R07666
R07999
R08283
R00758
R02454
R05831
R05701
R06562
R06917
R07140
R07667
R08083
R08306
R00868
R02455
R06894
R05702
R06563
R06918
R07141
R07703
R08084
R08307
R00870
R02555
R07133
R05703
R06570
R06928
R07142
R07705
R08085
R08379
R00875
R02703
R07134
R05812
R06571
R06931
R07146
R07747
R08086
R08380
R00880
R02896
R07135
R05823
R06621
R06935
R07147
R07751
R08087
R08383
R01093
R02928
R07143
R05827
R06734
R06941
R07148
R07759
R08094
R08385
R01095
R02983
R07144
R05879
R06784
R06980
R07156
R07761
R08096
R08442
R01158
R03183
R07145
Table S3. Cofactor compounds used in this study NADH
ATP
NAD+
ADP
H+
AMP
HCO3-
Formaldehyde
CO2
electron
H2O
Oxygen
NH3
Chloride
Orthophosphate
Nitrite
Pyrophosphate
Bromide
CoA
Fluoride
6|Page
Table S4. Synthetic Media (g/liter) ammonium acetate 2.2 biotin 0.00001 FeSO4·7H2O 0.01 glucose 20 K2HPO4·3H2O 0.5 KH2PO4 0.5 MgSO4 0.2 MnSO4·1H2O 0.01 NaCl 0.01 para-aminobenzoic acid 0.001
Table S5. EFF scores for novel reactants catalyzed by 4.2.1 enzymes with presence of crystal waters PDB
Ligand
EFF(Å2)
2ONE
3-Hydroxybutanal(R) 3-Hydroxybutanal(S) 3-Hydroxy-1-butanol(R) 3-Hydroxy-1-butanol(S)
5.037 5.091 4.274 3.334
7ACN 3-Hydroxybutanal(R) 3-Hydroxybutanal(S) 3-Hydroxy-1-butanol(R) 3-Hydroxy-1-butanol(S)
4.313 1.799 2.693 4.092
2DW7 3-Hydroxybutanal(R) 3-Hydroxybutanal(S) 3-Hydroxy-1-butanol(R) 3-Hydroxy-1-butanol(S)
1.342 1.964 0.005 0.125
7ENL
0.099 0.066 5.651 5.433
3-Hydroxybutanal(R) 3-Hydroxybutanal(S) 3-Hydroxy-1-butanol(R) 3-Hydroxy-1-butanol(S)
7|Page
Table S6. EFF scores for novel reactants catalyzed by 1.1.1 enzymes with presence of crystal waters PDB
Ligand
EFF(Å2)
PDB
Ligand
EFF(Å2)
1M2W
3-Hydroxybutanal(R) 3-Hydroxybutanal(S) Acetylacetaldehyde(a) Acetylacetaldehyde(b) 1-Hydroxy-3-butanone Crotonaldehyde
7.606 7.504 6.601 8.686 6.490 6.446
1MRQ
3-Hydroxybutanal(R) 3-Hydroxybutanal(S) Acetylacetaldehyde(a) Acetylacetaldehyde(b) 1-Hydroxy-3-butanone Crotonaldehyde
37.232 80.690 74.342 34.968 38.403 3.951
1PJ2
3-Hydroxybutanal(R) 3-Hydroxybutanal(S) Acetylacetaldehyde(a) Acetylacetaldehyde(b) 1-Hydroxy-3-butanone Crotonaldehyde
0.305 1.895 2.047 0.789 0.417 0.041
1KAE
3-Hydroxybutanal(R) 3-Hydroxybutanal(S) Acetylacetaldehyde(a) Acetylacetaldehyde(b) 1-Hydroxy-3-butanone Crotonaldehyde
6.593 9.060 66.625 49.166 5.859 70.867
3CXT
3-Hydroxybutanal(R) 3-Hydroxybutanal(S) Acetylacetaldehyde(a) Acetylacetaldehyde(b) 1-Hydroxy-3-butanone Crotonaldehyde
0.677 0.954 5.507 8.118 5.408 6.771
1PS6
3-Hydroxybutanal(R) 3-Hydroxybutanal(S) Acetylacetaldehyde(a) Acetylacetaldehyde(b) 1-Hydroxy-3-butanone Crotonaldehyde
23.644 23.807 22.257 21.066 25.528 28.788
3EEW
3-Hydroxybutanal(R) 3-Hydroxybutanal(S) Acetylacetaldehyde(a) Acetylacetaldehyde(b) 1-Hydroxy-3-butanone Crotonaldehyde
7.016 9.945 0.051 7.550 0.041 7.334
3KB6
3-Hydroxybutanal(R) 3-Hydroxybutanal(S) Acetylacetaldehyde(a) Acetylacetaldehyde(b) 1-Hydroxy-3-butanone Crotonaldehyde
2.772 0.741 5.158 0.655 0.167 15.545
8|Page
