Information on EC 1.1.1.268 - 2-(R)-hydroxypropyl-CoM dehydrogenase and Organism(s) Xanthobacter autotrophicus and UniProt Accession Q56840

-

-

r

(2R)-2-heptanol + NAD+

2-heptanone + NADH + H+

-

-

r

(2R)-2-hexanol + NAD+

2-hexanone + NADH + H+

-

-

r

(2R)-2-hydroxypropyl-CoM + NAD+

2-oxopropyl-CoM + NADH + H+

(2R)-2-octanol + NAD+

2-octanone + NADH + H+

-

-

r

(2R)-2-pentanol + NAD+

2-pentanone + NADH + H+

-

-

r

(2S)-2-butanol + NAD+

2-butanone + NADH + H+

very low activity

-

r

(2S)-2-hydroxypropyl-CoM + NAD+

2-oxopropyl-CoM + NADH + H+

competitive inhibitor to the R-enantiomer of substrate, very low activity

-

r

(2S)-2-pentanol + NAD+

2-pentanone + NADH + H+

-

-

r

(R)-2-butanol + NAD+

2-butanone + NADH + H+

-

-

r

(S)-2-butanol + NAD+

2-butanone + NADH + H+

-

-

r

2-(2-hydroxyethylthio)ethanesulfonate + NAD+

2-(formylmethylthio)ethanesulfonate + NADH + H+

achiral mimic of both R-hydroxypropyl-CoM and S-hydroxypropyl-CoM, substrate for both the R- and S-HPCDH enzymes with identical Km values

-

r

2-(2-hydroxyethylthio)ethanesulfonate + NADH + H+

?

very low activity

-

?

2-(R)-hydroxypropyl-CoM + NAD+

2-oxopropyl-CoM + NADH + H+

2-(S)-hydroxypropyl-CoM + NAD+

2-oxopropyl-CoM + NADH + H+

oxidation of S-hydroxypropyl-CoM with a kcat that is 402 times less than that for R-hydroxypropyl-CoM

-

r

2-butanone + NADH + H+

(2R)-2-butanol + NAD+

with no additives present, all forms of recombinant R-HPCDH prefer a re-face hydride addition to produce an enantiomeric excess (EE) of (S)-2-butanol, S- and R-2-butanol are comparably good substrates for the reverse reaction. The sulfonate of ethanesulfonate interacts with R152 and R196 in the CoM binding pocket alongside 2-butanone, in a way that discourages a si-face hydride addition to produce (R)-2-butanol

-

r

2-butanone + NADH + H+

(2S)-2-butanol + NAD+

with no additives present, all forms of recombinant R-HPCDH prefer a re-face hydride addition to produce an enantiomeric excess (EE) of (S)-2-butanol, S- and R-2-butanol are comparably good substrates for the reverse reaction. The sulfonate of ethanesulfonate interacts with R152 and R196 in the CoM binding pocket alongside 2-butanone, in a way that discourages a si-face hydride addition to produce (R)-2-butanol

-

r

2-butanone + NADH + H+

(S)-2-butanol + (R)-2-butanol + NAD+

-

without additions, 71.9% (S)-enantiomer + 28% (R)-enantiomer, in presence of 1 mM ethansulfonate 92.7% (S)-enantiomer + 7.3% (R)-enantiomer

-

r

2-oxopropyl-CoM + NADH + H+

2-(R)-hydroxypropyl-CoM + NAD+

-

-

r

2-propanol + NAD+

2-propanone + NADH + H+

-

-

r

(R)-2-butanol + NAD+

2-butanone + NADH + H+

-

the enantiomeric selectivity of the reverse reaction is 39.6% for (S)-2-butanol without additive, 90.8% with 1 mM CH3CH2SO3-Na+ as additive, 87.8% with 1 mM HSCH2CH2SO3-Na+ as additive, 52.0% with 1 mM CH3CH2COO-Na+ as additive, 46.6% with 1 mM CH3COO-Na+ as additive, 44.8% with 1 mM CH3CH2NH3+Cl- as additive, 45.4% with 1 mM CH3CH2OH as additive, 45.8% with 1 mM Na2SO4 as additive and 40.4% with 1 mM NaCl as additive

-

r

(R)-2-heptanol + NAD+

2-heptanone + NADH + H+

-

-

?

(R)-2-hexanol + NAD+

2-hexanone + NADH + H+

-

-

?

(R)-2-octanol + NAD+

2-octanone + NADH + H+

-

-

?

(R)-2-pentanol + NAD+

2-pentanone + NADH + H+

-

-

?

(S)-2-butanol + NAD+

2-butanone + NADH + H+

-

-

?

(S)-2-pentanol + NAD+

2-pentanone + NADH + H+

-

-

?

2-(R)-hydroxypropyl-CoM + NAD+

2-oxopropyl-CoM + NADH + H+

-

-

?

2-propanol + NAD+

acetone + NADH + H+

-

-

?

2-[(R)-2-hydroxypropylthio]ethanesulfonate + NAD+

2-(2-ketopropylthio)ethanesulfonate + NADH + H+

-

the enzyme is highly specific for the R-enantiomer

-

r

2-[(S)-2-hydroxypropylthio]ethanesulfonate + NAD+

2-(2-ketopropylthio)ethanesulfonate + NADH + H+

-

the enzyme is highly specific for the R-enantiomer

-

r

additional information

?

-

(2R)-2-hydroxypropyl-CoM + NAD+

2-oxopropyl-CoM + NADH + H+

-

-

r

2-(R)-hydroxypropyl-CoM + NAD+

2-oxopropyl-CoM + NADH + H+

-

-

?

NAD+

-

667740, 762714

NADH

-

NAD+

-

(2S)-2-hydroxypropyl-CoM

competitive inhibition

2-(2-methyl-2-hydroxypropylthio)-ethanesulfonate

M-HPC, competitive inhibition

-

2-(2-methyl-2-hydroxypropylthio)ethanesulfonate

competitive

2-bromoethanesulfonate

CoM-analogue, mixed type inhibitor. 32.7% residual activity at 5 mM, reversible inhibition

712458

CoM

mixed inhibition

ethanesulfonate

the addition of ethanesulfonate dramatically increases the enentiomeric excess of (S)-2-butanol produced by all forms of recombinant R-HPCDH except the R152A and R196A mutants. The effect of ethanesulfonate and other additives on (S)-butanol production is termed enantioselective modulation, and it is highly selective for sulfonate containing molecules. The interpretation is that the sulfonate of ethanesulfonate interacts with R152 and R196 in the CoM binding pocket alongside 2-butanone, in a way that discourages a si-face hydride addition to produce (R)-2-butanol

ethanesulfonate

-

1 mM, 5.6fold increase in ratio of turnover number to Km-value for 2-butanone as substrate

methanesulfonate

-

1 mM, 5.6fold increase in ratio of turnover number to Km-value for 2-butanone as substrate

propanesulfonate

-

1 mM, 1.7fold increase in ratio of turnover number to Km-value for 2-butanone as substrate

326

(2R)-2-butanol

recombinant enzyme, pH and temperature not specified in the publication

-

2.64

(2R)-2-heptanol

recombinant enzyme, pH and temperature not specified in the publication

-

4.6

(2R)-2-hexanol

recombinant enzyme, pH and temperature not specified in the publication

-

0.102

(2R)-2-hydroxypropyl-CoM

recombinant enzyme, pH and temperature not specified in the publication

-

1.08

(2R)-2-octanol

recombinant enzyme, pH and temperature not specified in the publication

-

20

(2R)-2-pentanol

recombinant enzyme, pH and temperature not specified in the publication

-

315

(2S)-2-butanol

recombinant enzyme, pH and temperature not specified in the publication

-

0.1

(2S)-2-hydroxypropyl-CoM

recombinant enzyme, pH and temperature not specified in the publication

153

(2S)-2-pentanol

recombinant enzyme, pH and temperature not specified in the publication

-

220

(R)-2-butanol

pH 7.5, 30°C

350

(S)-2-butanol

pH 7.5, 30°C

0.75 - 0.96

2-(2-hydroxyethylthio)ethanesulfonate

0.096 - 0.124

2-(R)-hydroxypropyl-CoM

0.22

2-(S)-hydroxypropyl-CoM

pH 7.5, 30°C

0.068

2-oxopropyl-CoM

pH 7.5, 30°C

1726

2-propanol

recombinant enzyme, pH and temperature not specified in the publication

328

(R)-2-butanol

-

pH 7.5, 30°C

2.64

(R)-2-heptanol

-

pH 7.5, 30°C

4.6

(R)-2-hexanol

-

pH 7.5, 30°C

1.08

(R)-2-octanol

-

pH 7.5, 30°C

20

(R)-2-pentanol

-

pH 7.5, 30°C

315

(S)-2-butanol

-

pH 7.5, 30°C

153

(S)-2-pentanol

-

pH 7.5, 30°C

0.092 - 0.44

2-(2-ketopropylthio)ethanesulfonate

2 - 84

2-butanone

5 entries

1726

2-propanol

-

pH 7.5, 30°C

0.102

2-[(R)-2-hydroxypropylthio]ethanesulfonate

-

pH 7.5, 30°C

0.1

2-[(S)-2-hydroxypropylthio]ethanesulfonate

-

pH 7.5, 30°C

additional information

steady-state kinetics, comparison of steady-state kinetics with wild-type an d mutant enzymes, overview

-

0.98

(2R)-2-butanol

recombinant enzyme, pH and temperature not specified in the publication

-

1.8

(2R)-2-heptanol

recombinant enzyme, pH and temperature not specified in the publication

-

2.7

(2R)-2-hexanol

recombinant enzyme, pH and temperature not specified in the publication

-

26.8

(2R)-2-hydroxypropyl-CoM

recombinant enzyme, pH and temperature not specified in the publication

-

1.7

(2R)-2-octanol

recombinant enzyme, pH and temperature not specified in the publication

-

1.1

(2R)-2-pentanol

recombinant enzyme, pH and temperature not specified in the publication

-

2.2

(2S)-2-butanol

recombinant enzyme, pH and temperature not specified in the publication

-

0.044

(2S)-2-hydroxypropyl-CoM

recombinant enzyme, pH and temperature not specified in the publication

2.9

(2S)-2-pentanol

recombinant enzyme, pH and temperature not specified in the publication

-

1.9

(R)-2-butanol

pH 7.5, 30°C

9.3

(S)-2-butanol

pH 7.5, 30°C

0.29 - 0.55

2-(2-hydroxyethylthio)ethanesulfonate

29 - 49

2-(R)-hydroxypropyl-CoM

0.12

2-(S)-hydroxypropyl-CoM

pH 7.5, 30°C

29

2-oxopropyl-CoM

pH 7.5, 30°C

2.9

2-propanol

recombinant enzyme, pH and temperature not specified in the publication

0.98

(R)-2-butanol

-

pH 7.5, 30°C

1.8

(R)-2-heptanol

-

pH 7.5, 30°C

2.7

(R)-2-hexanol

-

pH 7.5, 30°C

1.7

(R)-2-octanol

-

pH 7.5, 30°C

1.1

(R)-2-pentanol

-

pH 7.5, 30°C

2.2

(S)-2-butanol

-

pH 7.5, 30°C

2.9

(S)-2-pentanol

-

pH 7.5, 30°C

1.4 - 27.9

2-(2-ketopropylthio)ethanesulfonate

5 entries

0.012 - 0.033

2-butanone

5 entries

2.9

2-propanol

-

pH 7.5, 30°C

26.8

2-[(R)-2-hydroxypropylthio]ethanesulfonate

-

pH 7.5, 30°C

0.044

2-[(S)-2-hydroxypropylthio]ethanesulfonate

-

pH 7.5, 30°C

0.003

(2R)-2-butanol

recombinant enzyme, pH and temperature not specified in the publication

-

0.68

(2R)-2-heptanol

recombinant enzyme, pH and temperature not specified in the publication

-

0.59

(2R)-2-hexanol

recombinant enzyme, pH and temperature not specified in the publication

-

262.75

(2R)-2-hydroxypropyl-CoM

recombinant enzyme, pH and temperature not specified in the publication

-

1.57

(2R)-2-octanol

recombinant enzyme, pH and temperature not specified in the publication

-

0.055

(2R)-2-pentanol

recombinant enzyme, pH and temperature not specified in the publication

-

0.007

(2S)-2-butanol

recombinant enzyme, pH and temperature not specified in the publication

-

0.44

(2S)-2-hydroxypropyl-CoM

recombinant enzyme, pH and temperature not specified in the publication

0.019

(2S)-2-pentanol

recombinant enzyme, pH and temperature not specified in the publication

-

8.69

(R)-2-butanol

pH 7.5, 30°C

26.4

(S)-2-butanol

pH 7.5, 30°C

0.39

2-(2-hydroxyethylthio)ethanesulfonate

recombinant enzyme, pH and temperature not specified in the publication

500

2-(R)-hydroxypropyl-CoM

pH 7.5, 30°C

0.53

2-(S)-hydroxypropyl-CoM

pH 7.5, 30°C

420

2-oxopropyl-CoM

pH 7.5, 30°C

0.0017

2-propanol

recombinant enzyme, pH and temperature not specified in the publication

0.156

(2S)-2-hydroxypropyl-CoM

pH and temperature not specified in the publication, versus (2R)-2-hydroxypropyl-CoM

0.406

2-(2-methyl-2-hydroxypropylthio)-ethanesulfonate

pH and temperature not specified in the publication, versus (2R)-2-hydroxypropyl-CoM

-

0.29

2-(2-methyl-2-hydroxypropylthio)ethanesulfonate

pH 7.5, 30°C

1.2

2-bromoethanesulfonate

pH 7.4, 30°C

712458

2.25 - 3.62

CoM

evolution

the enzyme belongs to the short-chain dehydrogenases/reductase (SDR) superfamily of enzymes. The C-terminal domains of SDR enzymes are responsible for imparting substrate specificity

malfunction

substitution of R152 or R196 for alanine inhibits ethanesulfonate binding to the extent that its addition does not increase the EE of (S)-2-butanol produced by these mutants

metabolism

(R)- and (S)-hydroxypropyl-coenzyme M dehydrogenase (R- and S-HPCDH), are part of a bacterial pathway of short-chain alkene and epoxide metabolism. R- and S-HPCDH act on different substrate enantiomers in a common pathway

metabolism

-

the bacterium produces R- and S-HPCDH, EC 1.1.1.268 and EC 1.1.1.269, simultaneously to facilitate transformation of R- and S-enantiomers of epoxy-propane to a common achiral product 2-ketopropyl-CoM

additional information

tetramer

the tetramer is stabilized by the interaction of the terminal carboxylates of each subunit with divalent metal ions

667740

purified recombinant enzyme, X-ray diffraction structure determination and analysis

the enzyme is cocrystallized in the presence of (S)-hydroxypropyl-coenzyme M, structure refined to 1.8 A resolution

667740

R152A

site-directed mutagenesis, the mutant shows altered substrate specificity and interaction with additives compared to wild-type enzyme

R196A

site-directed mutagenesis, the mutant shows altered substrate specificity and interaction with additives compared to wild-type enzyme

R203A

site-directed mutagenesis, the mutant shows altered substrate specificity and interaction with additives compared to wild-type enzyme

R209A

site-directed mutagenesis, the mutant shows altered substrate specificity and interaction with additives compared to wild-type enzyme

R152A

-

the ratio of turnover number to Km-value for 2-(2-ketopropylthio)ethanesulfonate is 5192fold lower than the ratio for the wild-type enzyme. The ratio of turnover number to Km-value for 2-butanone is 1.3fold higher than the ratio for the wild-type enzyme

R179A

-

no detectable activity with 2-(2-ketopropylthio)ethanesulfonate and 2-butanone

R196A

-

the ratio of turnover number to Km-value for 2-(2-ketopropylthio)ethanesulfonate is 1000fold lower than the ratio for the wild-type enzyme. The ratio of turnover number to Km-value for 2-butanone is 2.7fold higher than the ratio for the wild-type enzyme

R203A

-

the ratio of turnover number to Km-value for 2-(2-ketopropylthio)ethanesulfonate is 4.3fold lower than the ratio for the wild-type enzyme. The ratio of turnover number to Km-value for 2-butanone is 1.6fold lower than the ratio for the wild-type enzyme

R209A

-

the ratio of turnover number to Km-value for 2-(2-ketopropylthio)ethanesulfonate is 2.7fold lower than the ratio for the wild-type enzyme. The ratio of turnover number to Km-value for 2-butanone is 1.4fold lower than the ratio for the wild-type enzyme

additional information

comparison of steady-state kinetics and 2-butanol production arte and stereochemistry of wild-type and mutant enzymes, overview

recombinant enzyme

expression in Escherichia coli

-

-

Clark, D.D.; Boyd, J.M.; Ensign, S.A.

The stereoselectivity and catalytic properties of Xanthobacter autotrophicus 2-[(R)-2-hydroxypropylthio]ethanesulfonate dehydrogenase are controlled by interactions between C-terminal arginine residues and the sulfonate of coenzyme M

Biochemistry

43

6763-6771

2004

Xanthobacter autotrophicus

15157110

667740

Krishnakumar, A.M.; Nocek, B.P.; Clark, D.D.; Ensign, S.A.; Peters, J.W.

Structural basis for stereoselectivity in the (R)- and (S)-hydroxypropylthioethanesulfonate dehydrogenases

Biochemistry

45

8831-8840

2006

Xanthobacter autotrophicus (Q56840)

16846226

Sliwa, D.A.; Krishnakumar, A.M.; Peters, J.W.; Ensign, S.A.

Molecular basis for enantioselectivity in the (R)- and (S)-hydroxypropylthioethanesulfonate dehydrogenases, a unique pair of stereoselective short-chain dehydrogenases/reductases involved in aliphatic epoxide carboxylation

Biochemistry

49

3487-3498

2010

Xanthobacter autotrophicus (Q56840)

20302306

712458

Boyd, J.M.; Clark, D.D.; Kofoed, M.A.; Ensign, S.A.

Mechanism of inhibition of aliphatic epoxide carboxylation by the coenzyme M analog 2-bromoethanesulfonate

J. Biol. Chem.

285

25232-25242

2010

Xanthobacter autotrophicus (Q56840)

20551308

Bakelar, J.W.; Sliwa, D.A.; Johnson, S.J.

Crystal structures of S-HPCDH reveal determinants of stereospecificity for R- and S-hydroxypropyl-coenzyme M dehydrogenases

Arch. Biochem. Biophys.

533

62-68

2013

Xanthobacter autotrophicus

23474457