An evaluation of the substrate specificity, and of its modification by site-directed mutagenesis, of the cloned L-lactate dehydrogenase from Bacillus stearothermophilus

Luyten, Marcel; Bur, Daniel; Wynn, Hla; Parris, Wendy; Gold, Marvin; Friesen, James D.; Jones, J. Bryan

doi:10.1021/ja00199a046

Cited by 34 publications

(20 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This suggests that there is an optimum amount of free space in the R group binding pocket consistent with about two methylene groups for the mutant enzymes. This conclusion is supported by molecular modeling, which also defines the active site as having a maximum capacity of one methylene group with Q102 and two methylene groups for both the Q102S and Q102T mutant enzymes and is in agreement with the results for the Q102N mutant BSLDH (Luyten et al, 1989).…”

Section: Straight Chain Aliphatic Substratessupporting

confidence: 84%

Substitution of the amino acid at position 102 with polar and aromatic residues influences substrate specificity of lactate dehydrogenase

Nicholls¹,

Davey²,

Jones³

et al. 1994

J Protein Chem

View full text Add to dashboard Cite

The Gln residue at amino acid position 102 of Bacillus stearothermophilus lactate dehydrogenase was replaced with Ser, Thr, Tyr, or Phe to investigate the effect on substrate recognition. The Q102S and Q102T mutant enzymes were found to have a broader range of substrate specificity (measured by kcat/Km) than the wild-type enzyme. However, it is evident that either Ser or Thr at position 102 are of a size able to accommodate a wide variety of substrates in the active site and substrate specificity appears to rely largely on size discrimination in these mutants. The Q102F and Q102Y mutant enzymes have low catalytic efficiency and do not show this relaxed substrate specificity. However, their activities are restored by the presence of an aromatic substrate. All of the enzymes have a very low catalytic efficiency with branched chain aliphatic substrates.

show abstract

Section: Straight Chain Aliphatic Substratessupporting

confidence: 84%

Substitution of the amino acid at position 102 with polar and aromatic residues influences substrate specificity of lactate dehydrogenase

Nicholls¹,

Davey²,

Jones³

et al. 1994

J Protein Chem

View full text Add to dashboard Cite

show abstract

“…In our model, this distance is 3.3 Å for quinate and 3.8 Å for shikimate. Furthermore, it is well known that in NAD(P) + /NAD(P)H-dependent dehydrogenases only a few residues are responsible for cosubstrate recognition, namely arginine and threonine or serine for NAD(P) + / NADPH-dependent enzymes and aspartate for NAD + / NADH-dependent proteins (see, for example, Luyten et al, 1989). The primary structure of CglQDH contains an Asp177-X-Asp179 motif.…”

Section: Potential Binding Site Of Nadhmentioning

confidence: 99%

1.6 Å structure of an NAD⁺-dependent quinate dehydrogenase fromCorynebacterium glutamicum

Schoepe

Niefind

Schomburg

2008

Acta Crystallogr D Biol Cryst

View full text Add to dashboard Cite

To date, three different functional classes of bacterial shikimate/quinate dehydrogenases have been identified and are referred to as AroE, SDH-L and YdiB. The enzyme AroE and the catalytically much slower SDH-L clearly prefer NADP+/NADPH as the cosubstrate and are specific for (dehydro-)shikimate, whereas in YdiB the differences in affinity for NADP+/NADPH versus NAD+/NADH as well as for (dehydro-)shikimate versus (dehydro-)quinate are marginal. These three subclasses have a similar three-dimensional fold and hence all belong to the same structural class of proteins. In this paper, the crystal structure of an enzyme from Corynebacterium glutamicum is presented that clearly prefers NAD+ as a cosubstrate and that demonstrates a higher catalytic efficiency for quinate rather than shikimate. While the kinetic constants for this enzyme clearly differ from those reported for AroE, SDH-L and YdiB, the three-dimensional structure of this protein is similar to members of these three subclasses. Thus, the enzyme described here belongs to a new functional class of the shikimate/quinate dehydrogenase family. The different substrate and cosubstrate specificities of this enzyme relative to all other known bacterial shikimate/quinate dehydrogenases are discussed by means of analyzing the crystal structure and derived models. It is proposed that in contrast to shikimate, quinate forms a hydrogen bond to the NAD+. In addition, it is suggested that the hydroxyl group of a conserved active-site threonine hydrogen bonds to quinate more effectively than to shikimate. Also, the hydroxyl group of a conserved tyrosine approaches the carboxylate group of quinate more closely than it does the carboxylate group of shikimate. Taken together, these factors most likely lead to a lower Michaelis constant and therefore to a higher catalytic efficiency for quinate. The active site of the dehydrogenase reported here is larger than those of other known shikimate/quinate dehydrogenases, which may explain why quinate is easily accommodated within the catalytic cleft.

show abstract

“…Mutagenesis. The BSLDH gene cloned into plasmid pTZ-R18 served as a source of single-stranded DNA for mutagenesis and sequencing as described previously (Luyten et al, 1989a).…”

Section: Methodsmentioning

confidence: 99%

Threonine 246 at the active site of the L-lactate dehydrogenase of Bacillus stearothermophilus is important for catalysis but not for substrate binding

Sakowicz

Kallwass²,

Parris³

et al. 1993

Biochemistry

Self Cite

View full text Add to dashboard Cite

Threonine 246 is an active site residue that is conserved in all known L-lactate dehydrogenase (LDH; EC 1.1.1.27) sequences. In order to investigate the role of Thr246 in Bacillus stearothermophilus LDH, this residue was altered by site-directed mutagenesis to valine, alanine, leucine, and serine, respectively. The effects of these mutations, as observed in both steady-state and single-turnover kinetic measurements with different substrates, demonstrated the importance for catalysis of a hydroxyl group in the 246 amino acid residue. In contrast, no significant contribution of the OH group of Thr246 to productive pyruvate binding was observed. Instead, it is proposed that the role of Thr246 may be to facilitate hydride transfer from the nicotinamide ring of the NADH cofactor to the pyruvate carbonyl group.

show abstract

An evaluation of the substrate specificity, and of its modification by site-directed mutagenesis, of the cloned L-lactate dehydrogenase from Bacillus stearothermophilus

Cited by 34 publications

References 0 publications

Substitution of the amino acid at position 102 with polar and aromatic residues influences substrate specificity of lactate dehydrogenase

Substitution of the amino acid at position 102 with polar and aromatic residues influences substrate specificity of lactate dehydrogenase

1.6 Å structure of an NAD⁺-dependent quinate dehydrogenase fromCorynebacterium glutamicum

Threonine 246 at the active site of the L-lactate dehydrogenase of Bacillus stearothermophilus is important for catalysis but not for substrate binding

Contact Info

Product

Resources

About

An evaluation of the substrate specificity, and of its modification by site-directed mutagenesis, of the cloned L-lactate dehydrogenase from Bacillus stearothermophilus

Cited by 34 publications

References 0 publications

Substitution of the amino acid at position 102 with polar and aromatic residues influences substrate specificity of lactate dehydrogenase

Substitution of the amino acid at position 102 with polar and aromatic residues influences substrate specificity of lactate dehydrogenase

1.6 Å structure of an NAD+-dependent quinate dehydrogenase fromCorynebacterium glutamicum

Threonine 246 at the active site of the L-lactate dehydrogenase of Bacillus stearothermophilus is important for catalysis but not for substrate binding

Contact Info

Product

Resources

About

1.6 Å structure of an NAD⁺-dependent quinate dehydrogenase fromCorynebacterium glutamicum