Crystal structure of a ternary complex of d-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei, NAD+ and 2-oxoisocaproate at 1.9 Å resolution (original) (raw)
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Journal of Molecular Biology, 1997
D-2-hydroxyisocaproate dehydrogenase (D-HicDH) from Lactobacillus casei is a homodimer with 333 amino acids and a molecular mass of 37 kDa per subunit. The enzyme belongs to the protein family of NAD-dependent D-2-hydroxycarboxylate dehydrogenases and within this family to the subgroup of D-lactate dehydrogenases (D-LDHs). Compared with other D-LDHs D-HicDH is characterized by a very low speci®city regarding size and chemical constitution of the accepted D-2-hydroxycarboxylates. Hexagonal crystals of recombinant D-HicDH in the presence of NAD and 2oxoisocaproate (4-methyl-2-oxopentanoate) were grown with ammonium sulphate as precipitating agent. The structure of these crystals was solved by molecular replacement and re®ned to a ®nal R-factor of 19.6% for all measured X-ray re¯ections in the resolution range (I to 1.86 A Ê). Both NAD and 2-oxoisocaproate were identi®ed in the electron density map; binding of the latter in the active site, however, competes with a sulphate ion, which is also de®ned by electron density. Additionally the ®nal model contains 182 water molecules and a second sulphate ion. The binding of both an in vitro substrate and the natural cosubstrate in the active site provides substantial insight into the catalytic mechanism and allows us to assess previously published active site models for this enzyme family, in particular the two most controversial points, the role of the conserved Arg234 and substrate binding. Furthermore the overall topology and details of the D-HicDH structure are described, discussed against the background of homologous structures and compared with one closely and one distantly related protein.
Journal of Molecular Biology, 1995
Gesellschaft fü r L-2-Hydroxyisocaproate dehydrogenase (L-HicDH) from Lactobacillus Biotechnologische Forschung confusus, a homotetramer with a molecular mass of 33 kDa per subunit, belongs to the protein family of the NAD +-dependent L-2-hydroxycarboxy-(GBF), Abteilung Molekulare Strukturforschung late dehydrogenases. L-HicDH was crystallized with ammonium sulphate as precipitant in the presence of NAD +. The crystals belong to the trigonal Mascheroder Weg 1, D-38124 space group P3 2 21, with a = 135.9 Å and c = 205.9 Å, and diffract X-rays to Braunschweig, Germany 2.2 Å resolution. The crystal structure was solved by Patterson search and molecular replacement techniques and refined to an R-value of 21.4 % (2.2 to 8 Å). The final structure model contains one NAD + molecule and one sulphate ion per subunit, with 309 water molecules. An unusual feature of this crystal structure is the deviation of the protein subunits from non-crystallographic symmetry, which is so strong that it can be detected globally by self-rotation calculations in reciprocal space. This asymmetry is especially pronounced in the environment of the active site; it is reflected also in the nicotinamide conformation of NAD + and allows some conclusions to be drawn about the catalytic mechanism. In this context, an ''inner active site loop'' is identified as a structural element of fundamental functional importance. Furthermore, with knowledge of the crystal structure of L-HicDH the differences in substrate specificity between L-HicDH and the L-lactate dehydrogenases can be partly explained.
Refined crystal structure of dogfish M4 apo-lactate dehydrogenase
Journal of Molecular Biology, 1987
The crystal structure of M, apo-lactate dehydrogenase from the spiny dogfish (Squalus acanthius) was initially refined by a constrained-restrained, and subsequently restrained, least-squares technique. The final structure contained 286 water molecules and two sulfate ions per subunit and gave an R-factor of 0.202 for difraction data between 8-O and 2.0 A resolut,ion. The upper limit for the co-ordinate accuracy of the atoms was estimated t,o be 0.25 A.
Structure-Function Relationships in Lactate Dehydrogenase
Proceedings of the National Academy of Sciences, 1973
The binding of coenzyme and substrate are considered in relation to the known primary and tertiary structure of lactate dehydrogenase ). The adenine binds in a hydrophobic crevice, and the two coenzyme phosphates are oriented by interactions with the protein. The positively charged guanidinium group of argi-
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2009
The crystal structure of d-lactate dehydrogenase from Aquifex aeolicus (aq_727) was determined to 2.12 Å resolution in space group P2 1 2 1 2 1 , with unit-cell parameters a = 90.94, b = 94.43, c = 188.85 Å. The structure was solved by molecular replacement using the coenzyme-binding domain of Lactobacillus helveticus d-lactate dehydrogenase and contained two homodimers in the asymmetric unit. Each subunit of the homodimer was found to be in a 'closed' conformation with the NADH cofactor bound to the coenzyme-binding domain and with a lactate (or pyruvate) molecule bound at the interdomain active-site cleft.
Deletion variants of l-hydroxyisocaproate dehydrogenase. Probing substrate specificity
European Journal of Biochemistry, 1994
The substrate specificity and catalytic activity of the dinucleotide-dependent L-2-hydroxyisocaproate dehydrogenase from Lactobacillus confisus (L-HicDH) have been altered by modifying an enzyme region which is assumed to be involved in substrate recognition. The design of the variant enzymes was based on an amino acid alignment of the modified region with the functionally related L-lactate dehydrogenases. The best absolute sequence similarity for a protein with known tertiary structure was found for L-lactate dehydrogenase from dogfish (23%). In this study, the coenzyme loop, a functional element which is essential for catalysis and substrate specificity, was modified in order to identify the residues involved in the catalytic reaction and observe the effect on the substrate specificity. Deletions were introduced into the L-hydroxyisocaproate gene by site-directed mutagenesis. Several deletion-variant enzymes IlelOOAd, LyslOOBd, LeulOld, Asnl05Ad and ProlOSBd showed an altered substrate specificity. For the variant enzyme with the deletion of Asn/ProlOSA/ B, 2-0x0 carboxylic acids branched at C4 proved to be better substrates than 2-oxocaproate, the substrate with the best kc,/KM ratio known for the wild-type enzyme. The mutation resulted in a 5.2-fold increased catalytic efficiency towards 2-oxoisocaproate compared to the wild-type enzyme. After deleting Ile/LyslOOA/B, 2-phenylpyruvate is the only substrate which is still converted at a significant catalytic rate. The k,,, ratios of 2-oxocaproate versus 2-phenylpyruvate changed by a factor of 6500 when comparing wild-type enzyme and deletion-variant enzyme data. The single amino acid deletions in position lOOA and 100B caused drastic reductions in the catalytic activity for all tested substrates, whereas the deletion of LyslOOB, LeulO1, AsnlOSA as well as ProlOSB showed more specific modifications in catalytic rates and substrate recognition for each tested substrate.
Journal of Biological Chemistry, 2005
The three-dimensional structures of NAD-dependent D-lactate dehydrogenase (D-LDH) and formate dehydrogenase (FDH), which resemble each other, imply that the two enzymes commonly employ certain main chain atoms, which are located on corresponding loop structures in the active sites of the two enzymes, for their respective catalytic functions. These active site loops adopt different conformations in the two enzymes, a difference likely attributable to hydrogen bonds with Asn 97 and Glu 141 , which are also located at equivalent positions in D-LDH and FDH, respectively. X-ray crystallography at 2.4-Å resolution revealed that replacement of Asn 97 with Asp did not markedly change the overall protein structure but markedly perturbed the conformation of the active site loop in Lactobacillus pentosus D-LDH. The Asn 97 3 Asp mutant D-LDH exhibited virtually the same k cat , but about 70-fold higher K M value for pyruvate than the wild-type enzyme. For Paracoccus sp. 12-A FDH, in contrast, replacement of Glu 141 with Gln and Asn induced only 5.5-and 4.3-fold increases in the K M value, but 110 and 590-fold decreases in the k cat values for formate, respectively. Furthermore, these mutant FDHs, particularly the Glu 141 3 Asn enzyme, exhibited markedly enhanced catalytic activity for glyoxylate reduction, indicating that FDH is converted to a 2-hydroxy-acid dehydrogenase on the replacement of Glu 141 . These results indicate that the active site loops play different roles in the catalytic reactions of D-LDH and FDH, stabilization of substrate binding and promotion of hydrogen transfer, respectively, and that Asn 97 and Glu 141 , which stabilize suitable loop conformations, are essential elements for proper loop functioning.