Literature summary for 1.1.1.B20 extracted from

Subramanian, V.; Lunin, V.V.; Farmer, S.J.; Alahuhta, M.; Moore, K.T.; Ho, A.; Chaudhari, Y.B.; Zhang, M.; Himmel, M.E.; Decker, S.R.
Phylogenetics-based identification and characterization of a superior 2,3-butanediol dehydrogenase for Zymomonas mobilis expression (2020), Biotechnol. Biofuels, 13, 186 .

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Cloned(Commentary)

Cloned (Comment)	Organism
gene budC, sequence comparisons and phylogenetic analysis, recombinant expression in Zymomonas mobilis. Crude extracts from Zymomonas mobilis expressing Serratia marcescens Bdh (SmBdh) have the highest activity (8.89 micromol/min/mg), when compared to other Bdh enzymes (0.34-2.87 micromol/min/mg), overview	Serratia marcescens

Crystallization (Commentary)

Crystallization (Comment)	Organism
recombinant wild-type and mutant SmBdh proteins in complex with NAD+ and acetoin both bound in the active site, sitting drop vapor diffusion method, protein solution for wild-type SmBdh contains 9 mg/ml of protein, 20 mM Tris, pH 7.5, 100 mM NaCl, 20 mM NAD+, and 200 mM acetoin, the protein solution for mutant SmBdh Q247A consists of 4.2 mg/ml of protein, 20 mM Tris, pH 7.5, 100 mM NaCl, 20 mM NAD+, and 20 mM acetoin, and for mutant SmBdh Q247V/V139Q of 14.5 mg/ml of protein, 20 mM Tris, pH 7.5, 100 mM NaCl, 20 mM NAD+, and 20 mM acetoin, the well solution contains 0.1 M sodium malonate, pH 6-7 and 6-15% w/v PEG 3350, mixing of 200 nl of protein with 100-300 nl of well solution, equilibration against 0.05 ml of well solution, 20°C, X-ray diffraction structure determination and analysis at 1.8-2.0 A resolution	Serratia marcescens
recombinant wild-type and mutant SmBdh proteins in complex with with NAD+ and acetoin both bound in the active site, sitting drop vapor diffusion method, protein solution for wild-type SmBdh contains 9 mg/ml of protein, 20 mM Tris, pH 7.5, 100 mM NaCl, 20 mM NAD+, and 200 mM acetoin, the protein solution for mutant SmBdh Q247A consists of 4.2 mg/ml of protein, 20 mM Tris, pH 7.5, 100 mM NaCl, 20 mM NAD+, and 20 mM acetoin, and for mutant SmBdh Q247V/V139Q of 14.5 mg/ml of protein, 20 mM Tris, pH 7.5, 100 mM NaCl, 20 mM NAD+, and 20 mM acetoin, the well solution contains 0.1 M sodium malonate, pH 6-7 and 6-15% w/v PEG 3350, mixing of 200 nl of protein with 100-300 nl of well solution, equilibration against 0.05 ml of well solution, 20°C, X-ray diffraction structure determination and analysis at 1.8-2.0 A resolution	Serratia marcescens

Crystallization (Comment)

Organism

recombinant wild-type and mutant SmBdh proteins in complex with NAD+ and acetoin both bound in the active site, sitting drop vapor diffusion method, protein solution for wild-type SmBdh contains 9 mg/ml of protein, 20 mM Tris, pH 7.5, 100 mM NaCl, 20 mM NAD+, and 200 mM acetoin, the protein solution for mutant SmBdh Q247A consists of 4.2 mg/ml of protein, 20 mM Tris, pH 7.5, 100 mM NaCl, 20 mM NAD+, and 20 mM acetoin, and for mutant SmBdh Q247V/V139Q of 14.5 mg/ml of protein, 20 mM Tris, pH 7.5, 100 mM NaCl, 20 mM NAD+, and 20 mM acetoin, the well solution contains 0.1 M sodium malonate, pH 6-7 and 6-15% w/v PEG 3350, mixing of 200 nl of protein with 100-300 nl of well solution, equilibration against 0.05 ml of well solution, 20°C, X-ray diffraction structure determination and analysis at 1.8-2.0 A resolution

Serratia marcescens

recombinant wild-type and mutant SmBdh proteins in complex with with NAD+ and acetoin both bound in the active site, sitting drop vapor diffusion method, protein solution for wild-type SmBdh contains 9 mg/ml of protein, 20 mM Tris, pH 7.5, 100 mM NaCl, 20 mM NAD+, and 200 mM acetoin, the protein solution for mutant SmBdh Q247A consists of 4.2 mg/ml of protein, 20 mM Tris, pH 7.5, 100 mM NaCl, 20 mM NAD+, and 20 mM acetoin, and for mutant SmBdh Q247V/V139Q of 14.5 mg/ml of protein, 20 mM Tris, pH 7.5, 100 mM NaCl, 20 mM NAD+, and 20 mM acetoin, the well solution contains 0.1 M sodium malonate, pH 6-7 and 6-15% w/v PEG 3350, mixing of 200 nl of protein with 100-300 nl of well solution, equilibration against 0.05 ml of well solution, 20°C, X-ray diffraction structure determination and analysis at 1.8-2.0 A resolution

Serratia marcescens

Protein Variants

Protein Variants	Comment	Organism
additional information	enzyme SmBdh is superior to other Bdhs for expression in Zymomonas mobilis for 2,3-BDO production. Structurally guided changes of recombinantly SmBdh expressed in Zymomonas mobilis can explain its superiority over lower activity Bdh enzymes from the same family of proteins. Development of two mutants of SmBdh: (1) Q247A where the Gln247 side chain is removed, leaving alanine at position 247 and (2) the double mutant Q247A/V139Q, where the missing glutamine side chain is reinstated at the position 139 that is present in KpBdh. Whereas Q247A will disrupt the active site of the protein, Q247A/V139Q is expected to restore the active site via a compensatory mechanism resulting in the active site being established by a single protein chain without contribution from a symmetry-related molecule (i.e. from the C-terminus of the opposite molecule in the tetramer)	Serratia marcescens
Q274A	site-directed mutagenesis, the substrate binding site is occupied by a glycerol molecule in the Q247A mutant, the mutation disrupts the active site of the protein, the Q247A mutant shows a 90% loss in activity compare to wild-type	Serratia marcescens
Q274A/V139Q	site-directed mutagenesis, the substrate binding site is occupied by an ethylene glycol molecule in the Q274A/V139Q mutant, the mutation disrupts the active site of the protein, the double mutant Q247A/V139Q showa 300% improvement in activity in comparison to the Q247A mutant. Although the double mutant does not completely restore the loss of Gln247 activity, significant function is regained by introducing the V139Q mutation in this protein, to about 50% activity compared to wild-type	Serratia marcescens

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
(2R,3S)-butane-2,3-diol + NAD+	Serratia marcescens	-	(3R)-acetoin + NADH + H+	-	r
(2R,3S)-butane-2,3-diol + NAD+	Serratia marcescens 9C	-	(3R)-acetoin + NADH + H+	-	r
(2S,3S)-butane-2,3-diol + NAD+	Serratia marcescens	-	(S)-acetoin + NADH + H+	-	r
(2S,3S)-butane-2,3-diol + NAD+	Serratia marcescens 9C	-	(S)-acetoin + NADH + H+	-	r

Organism

Organism	UniProt	Comment	Textmining
Serratia marcescens	H9XP47	-	-
Serratia marcescens	H9XP47	a ZM4 derivative strain with xylose-utilizing abilities	-
Serratia marcescens 9C	H9XP47	a ZM4 derivative strain with xylose-utilizing abilities	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
(2R,3S)-butane-2,3-diol + NAD+	-	Serratia marcescens	(3R)-acetoin + NADH + H+	-	r
(2R,3S)-butane-2,3-diol + NAD+	-	Serratia marcescens 9C	(3R)-acetoin + NADH + H+	-	r
(2S,3S)-butane-2,3-diol + NAD+	-	Serratia marcescens	(S)-acetoin + NADH + H+	-	r
(2S,3S)-butane-2,3-diol + NAD+	-	Serratia marcescens 9C	(S)-acetoin + NADH + H+	-	r
additional information	substrate promiscuity of the SmBdh enzyme, SmBdh has been reported to be able to reduce both (3R)- and (3S)-acetoin to 2,3-BDO, although (3R)-acetoin is more readily converted than (3S)-acetoin. On the other hand, SmBdh is able to oxidize meso-2,3-BDO and (2S,3S)-BDO, while oxidation of (2R,3R)-BDO is not detectable. Moreover, its activity towards (2S,3S)-BDO is only 11% of that towards meso-2,3-BDO. The enzyme is classified as an S-acting Bdh based on production of meso-2,3-BDO and (2S,3S)-BDO from a racemic mixture of acetoin	Serratia marcescens	?	-	-
additional information	substrate promiscuity of the SmBdh enzyme, SmBdh has been reported to be able to reduce both (3R)- and (3S)-acetoin to 2,3-BDO, although (3R)-acetoin is more readily converted than (3S)-acetoin. On the other hand, SmBdh is able to oxidize meso-2,3-BDO and (2S,3S)-BDO, while oxidation of (2R,3R)-BDO is not detectable. Moreover, its activity towards (2S,3S)-BDO is only 11% of that towards meso-2,3-BDO. The enzyme is classified as an S-acting Bdh based on production of meso-2,3-BDO and (2S,3S)-BDO from a racemic mixture of acetoin	Serratia marcescens 9C	?	-	-

Subunits

Subunits	Comment	Organism
tetramer	-	Serratia marcescens

Synonyms

Synonyms	Comment	Organism
2,3-butanediol dehydrogenase	-	Serratia marcescens
BDH	-	Serratia marcescens
budC	-	Serratia marcescens
SmBdh	-	Serratia marcescens

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7	-	assay at	Serratia marcescens

Cofactor

Cofactor	Comment	Organism	Structure
NAD+	enzyme binding structure, overview	Serratia marcescens
NADH	-	Serratia marcescens

General Information

General Information	Comment	Organism
evolution	Bdh enzymes can be classified into R-acting or S-acting depending on the chirality of the chiral center introduced by the enzyme at the acetoin C2 atom. Whereas the preference for (3R)-acetoin or (3S)-acetoin is imprinted in the geometry of the substrate-binding pocket, R-acting and S-acting Bdh enzymes belong to different protein families and possess different architectures	Serratia marcescens
malfunction	extending the alpha6 helix of SmBdh to mimic the lower activity Enterobacter cloacae enzyme EcBdh results in reduction of SmBdh function to nearly 3% of the total activity. In great contrast, reduction of the corresponding alpha6 helix of the EcBdh to mimic the SmBdh structure results in about 70% increase in its activity	Serratia marcescens
additional information	SmBdh shows a more extensive supporting hydrogen-bond network in comparison to the other well-studied Bdh enzymes, which enables improved substrate positioning and substrate specificity. The substrate-binding pocket is formed by two protein molecules, not a single peptide as found in all other reported Bdh enzymes. The C-terminus of molecule A protrudes into the groove between alpha7 helix and the alpha-turn alphat1 capping substrate-binding pocket of molecule Asymm and vice versa. The SmBdh active site is populated by a Gln247 residue contributed by the diagonally opposite subunit. The enzyme protein also contains a short alpha6 helix, which provides more efficient entry and exit of molecules from the active site, thereby contributing to enhanced substrate turnover. While coordinated active site formation is a unique structural characteristic of this tetrameric complex, the smaller alpha6 helix and extended hydrogen network contribute towards improved activity and substrate promiscuity of the enzyme. Gln247 plays a crucial role in SmBdh catalysis	Serratia marcescens