NAD+-Dependent d-2-Hydroxyisocaproate Dehydrogenase of Lactobacillus Delbrueckii subsp. Bulgaricus. Gene Cloning and Enzyme Characterization (original) (raw)
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Journal of Bacteriology, 2009
Hydroxyacid dehydrogenases of lactic acid bacteria, which catalyze the stereospecific reduction of branchedchain 2-keto acids to 2-hydroxyacids, are of interest in a variety of fields, including cheese flavor formation via amino acid catabolism. In this study, we used both targeted and random mutagenesis to identify the genes responsible for the reduction of 2-keto acids derived from amino acids in Lactococcus lactis. The gene panE, whose inactivation suppressed hydroxyisocaproate dehydrogenase activity, was cloned and overexpressed in Escherichia coli, and the recombinant His-tagged fusion protein was purified and characterized. The gene annotated panE was the sole gene responsible for the reduction of the 2-keto acids derived from leucine, isoleucine, and valine, while ldh, encoding L-lactate dehydrogenase, was responsible for the reduction of the 2-keto acids derived from phenylalanine and methionine. The kinetic parameters of the His-tagged PanE showed the highest catalytic efficiencies with 2-ketoisocaproate, 2-ketomethylvalerate, 2-ketoisovalerate, and benzoylformate (V max /K m ratios of 6,640, 4,180, 3,300, and 2,050 U/mg/mM, respectively), with NADH as the exclusive coenzyme. For the reverse reaction, the enzyme accepted D-2-hydroxyacids but not L-2-hydroxyacids. Although PanE showed the highest degrees of identity to putative NADP-dependent 2-ketopantoate reductases (KPRs), it did not exhibit KPR activity. Sequence homology analysis revealed that, together with the Dmandelate dehydrogenase of Enterococcus faecium and probably other putative KPRs, PanE belongs to a new family of D-2-hydroxyacid dehydrogenases which is unrelated to the well-described D-2-hydroxyisocaproate dehydrogenase family. Its probable physiological role is to regenerate the NAD ؉ necessary to catabolize branched-chain amino acids, leading to the production of ATP and aroma compounds.
European Journal of Biochemistry, 1997
The steady-state kinetics of D-2-hydroxy-4-methylvalerate dehydrogenase have been studied at pH 8.0 by initial velocity, product inhibition, and dead-end inhibition techniques. The mechanism is rapid-equilibrium ordered in the NAD' plus D-2-hydroxy-4-methylvalerate direction, and steady-state ordered in the other direction. In both cases coenzyme is the first substrate added and both the E-NADH-~-Zhydroxy-4-methylvalerate and E-NAD' -2-0x0-4-methylvalerate give rise to abortive complexes which cause excess substrate inhibition. Steady-state measurements show that the rate-limiting step in both directions at pH 8.0 is between formation of the enzyme-coenzyme-substrate ternary complex and the release of the first product of the reaction. Transient kinetics combined with primary kinetic deuterium isotope effects show that in the NADH -NAD' direction there is a slow, rate-limiting rearrangement of the E-NADHoxoacid complex while hydride transfer is very fast. The release of NAD' at pH 8.0 is 200-times faster than k,,, (NADH -NAD') whereas the release of NADH is only 5-times faster than k,,, (NAD' -NADH). The pH dependence of NADH binding depends upon the presence of two ionizable residues with a pK, of about 5.9. The pH dependence of kinetic parameters is explained by a third ionizable residue with pK, values 7.2 (in the E-NADH complex) and c6.4 (in the E-NAD' complex) which may be the proton donor and acceptor for the chemical reaction.
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.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2008
A putative Type II NADH dehydrogenase from Halobacillus dabanensis was recently reported to have Na + /H + antiport activity (and called Nap), raising the possibility of direct coupling of respiration to antiport-dependent pH homeostasis. This study characterized a homologous type II NADH dehydrogenase of genetically tractable alkaliphilic Bacillus pseudofirmus OF4, in which evidence supports antiport-based pH homeostasis that is mediated entirely by secondary antiport. Two candidate type II NADH dehydrogenase genes with canonical GXGXXG motifs were identified in a draft genome sequence of B. pseudofirmus OF4. The gene product designated NDH-2A exhibited homology to enzymes from Bacillus subtilis and Escherichia coli whereas NDH-2B exhibited homology to the H. dabanensis Nap protein and its alkaliphilic Bacillus halodurans C-125 homologue. The ndh-2A, but not the ndh-2B, gene complemented the growth defect of an NADH dehydrogenase-deficient E. coli mutant. Neither gene conferred Na + -resistance on an antiporterdeficient E. coli strain, nor did they confer Na + /H + antiport activity in vesicle assays. The purified hexahistidine-tagged gene products were approximately 50 kDa, contained noncovalently bound FAD and oxidized NADH. They were predominantly cytoplasmic in E. coli, consonant with the absence of antiport activity. The catalytic properties of NDH-2A were more consistent with a major respiratory role than those of NDH-2B. . DNA sequences: new sequence for Bp ndh-2A and ndh-2B were deposited Genbank accession no. EU030627 and EU030628
Purification and properties of NADH dehydrogenase from an alkalophilic bacillus
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1983
Sulfolobus acidocaldarius, a thermoacidophilic archaebacterium optimally growing at pH 2-3 and 75°C. A 2,100-fold purification was achieved. The purified enzyme is an acidic protein with an isoelectric point of 5.6 and a molecular weight of 95,000, consisting of two 50,000-dalton subunits. The enzyme showed an absorption spectrum characteristic of flavoproteins, with maxima at 272, 372, and 448 nm. The enzyme is highly thermostable, is specific for NADH as an electron donor, and is capable of using 2,6-dichlorophenolindophenol, ferricyanide, benzoquinone, and naphthoquinone as electron acceptors. Though at a low rate, caldariellaquinone, a unique and sole benzothiophenequinone in the genus Sulfolobus, was also reduced by the enzyme, suggesting that the enzyme is a possible member of the respiratory chain of the thermoacidophilic archaebacterium.
FEBS Letters, 1991
A straiti of Escherichiu coli (FMJ144) deficient for pyruvate formate lyase and lactate dehydrogenase (LWH) was complemented with a genomic DNA iibc\lry from Laciobacilius delbrueckii subsp. Lirlgaricus. One positive clone showed LWH activity and production of W(-)lactate was demonstrated,. The nucleotide sequence of the W-LDH gene (/&A) revealed the spontaneous inseetion of an E. coli insertion sequence IS2 upstream OF the gene cod&g region. The open reading frame encoded a 333-amino acid protein, showing no similarity with known L-LWH sequences but closely related to L. cosei W-hydroxyisocaproate dehydrogenase (W-HicDW). W-Lactate dehydtogenase; Ins&on sequence 1st; Lactobacillus delbrueckii subsp. bulgaricus; W-Hydroxyisocapeoate dehydrogenase Published by Elsevier Science Publishers B. V.
Biotechnology Letters, 2018
Objectives To identify a robust NADP ? dependent formate dehydrogenase from Lactobacillus buchneri NRRL B-30929 (LbFDH) with unique biochemical properties. Results A new NADP ? dependent formate dehydrogenase gene (fdh) was cloned from genomic DNA of L. buchneri NRRL B-30929. The recombinant construct was expressed in Escherichia coli BL21(DE3) with 6 9 histidine at the C-terminus and the purified protein obtained as a single band of approx. 44 kDa on SDS-PAGE and 90 kDa on native-PAGE. The LbFDH was highly active at acidic conditions (pH 4.8-6.2). Its optimum temperature was 60°C and 50°C with NADP ? and NAD ? , respectively and its T m value was 78°C. Its activity did not decrease after incubation in a solution containing 20% of DMSO and acetonitrile for 6 h. The K M constants were 49.8, 0.12 and 1.68 mM for formate (with NADP ?), NADP ? and NAD ? , respectively. Conclusions An NADP ? dependent FDH from L. buchneri NRRL B-30929 was cloned, expressed and identified with its unusual characteristics. The LbFDH can be a promising candidate for NADPH regeneration Electronic supplementary material The online version of this article (
1991
Southern blot analysis indicated that the gene encoding the constitutive, NADP-linked bile acid 7a-hydroxysteroid dehydrogenase of Eubacterium sp. strain VPI 12708 was located on a 6.5-kb EcoRI fragment of the chromosomal DNA. This fragment was cloned into bacteriophage lambda gtll, and a 2.9-kb piece of this insert was subcloned into pUC19, yielding the recombinant plasmid pBH51. DNA sequence analysis of the 7a-hydroxysteroid dehydrogenase gene in pBH51 revealed a 798-bp open reading frame, coding for a protein with a calculated molecular weight of 28,500. A putative promoter sequence and ribosome binding site were identified. The 7a-hydroxysteroid dehydrogenase mRNA transcript in Eubacterium sp. strain VPI 12708 was about 0.94 kb in length, suggesting that it is monocistronic. An Escherichia coli DH5a transformant harboring pBH51 had approximately 30-fold greater levels of 7a-hydroxysteroid dehydrogenase mRNA, immunoreactive protein, and specific activity than Eubacterium sp. strain VPI 12708. The 7af-hydroxysteroid dehydrogenase purified from the pBH51 transformant was similar in subunit molecular weight, specific activity, and kinetic properties to that from Eubacterium sp. strain VPI 12708, and it reacted with antiserum raised against the authentic enzyme on Western immunoblots. Alignment of the amino acid sequence of the 7a-hydroxysteroid dehydrogenase with those of 10 other pyridine nucleotide-linked alcohol/polyol dehydrogenases revealed six conserved amino acid residues in the N-terminal regions thought to function in coenzyme binding.
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.