Distinct conformation-mediated functions of an active site loop in the catalytic reactions of NAD-dependent D-lactate dehydrogenase and formate dehydrogenase

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 Asn97 and Glu141, which are also located at equivalent positions in D-LDH and FDH, respectively. X-ray crystallography at 2.4-A resolution revealed that replacement of Asn97 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 Asn97-->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 Glu141 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 Glu141-->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 Glu141. 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 Asn97 and Glu141, which stabilize suitable loop conformations, are essential elements for proper loop functioning.     

Biotransformation of R-2-hydroxy-4-phenylbutyric acid by D-lactate dehydrogenase and Candida boidinii cells containing formate dehydrogenase coimmobilized in a fibrous bed bioreactor

R-2-hydroxy-4-phenylbutyric acid (R-HPBA) is an important intermediate in the manufacture of angiotensin converting enzyme inhibitors. In this work, a recombinant D-lactate dehydrogenase (LDH) was used to transform 2-oxo-4-phenylbutyric acid (OPBA) to R-HPBA, with concomitant oxidation of beta-nicotinamide adenine dinucleotide (NADH) to NAD(+). The cofactor NADH was regenerated by formate dehydrogenase (FDH) present in whole cells of Candida boidinii, which were pre-treated with toluene to make them permeable. The whole cells used in the process were more stable and easier to prepare as compared with the isolated FDH from the cells. Kinetic study showed that the reaction rate was dependent on the concentration of cofactor, NAD(+), and that both R-HPBA and OPBA inhibited the reaction. A novel method for co-immobilization of whole cells and LDH enzyme on cotton cloth was developed using polyethyleneimine (PEI), which induced the formation of PEI-enzyme-cell aggregates and their adsorption onto cotton cloth, leading to multilayer co-immobilization of cells and enzyme with high loading (0.5 g cell and 8 mg LDH per gram of cotton cloth) and activity yield ( > 95%). A fibrous bed bioreactor with co-immobilized cells and enzyme on the cotton cloth was then evaluated for R-HPBA production in fed-batch and repeated batch modes, which gave relatively stable reactor productivity of 9 g/L . h and product yield of 0.95 mol/mol OPBA when the concentrations of OPBA and R-HPBA were less than 10 g/L.     

Stereospecific synthesis of (R)-2-hydroxy carboxylic acids using recombinant E. coli BL21 overexpressing YiaE from Escherichia coli K12 and glucose dehydrogenase from Bacillus subtilis

The yiaE gene from Escherichia coli K12 was functionally expressed in E. coli BL21 using an IPTG inducible pET expression system (2.1 U/mg), and YiaE was purified to a specific activity of 18 U/mg. The purified enzyme catalyzes reduction of various aromatic and aliphatic 2-oxo carboxylic acids to the corresponding (R)-2-hydoxy carboxylic acids using NADPH. For practical applications, the problem of NADPH recycle was effectively solved by using recombinant E. coli overexpressing YiaE and glucose dehydrogenase from Bacillus subtilis in the same cell. The recombinant E. coli was used to prepare (R)-phenyllactic acid and (R)-2-hydroxy-4-phenylbutanoic acid from the corresponding 2-oxo carboxylic acids (98% ee) while the alpha-carbonyl group of 2,4-dioxo-4-phenylbutyric acid was reduced regio- and stereospecifically to give (R)-2-hydroxy-4-oxo-4-phenylbutyric acid (97% ee) in quantitative yields. The cells could be recycled for 3 days at room temperature in 100 mM phosphate buffer (pH 7.0) without loss of activity, which reduced to 70% after 1 week.     

Designs for a broad substrate specificity keto acid dehydrogenase

Variations have been made to the structure of the nicotinamide adenine dinucleotide (NAD) dependent L-lactate dehydrogenase from Bacillus stearothermophilus at regions of the enzyme that we believe determine specificity toward different alpha-hydroxy acids (RCHOHCOO-, R = CH3, C2H5, etc.). Two regions of LDH that border the active site (but are not involved in the catalytic reaction) were altered in order to accommodate substrates with hydrophobic side chains larger than that of the naturally preferred substrate, pyruvate (R = CH3). The mutations 102-105GlnLysPro----MetValSer and 236-237AlaAla----GlyGly were made to increase the tolerance for large hydrophobic substrate side chains. The triple and double mutants alone gave little improvement for branched-chain-substituted pyruvates. The five changes together produced a broader substrate specificity alpha-hydroxy acid dehydrogenase, with a 55-fold improved kcat for alpha-ketoisocaproate to a value about 1/14 that of the native enzyme for pyruvate. Rational protein engineering enabled coupled changes in enzyme structure to be obtained with greater probability of success than random mutagenesis.     

Continuous enzymatic transformation in an enzyme membrane reactor with simultaneous NAD(H) regeneration. Reprinted from Biotechnology and Bioengineering, Vol. XXIII, No. 12, Pages 2789-2802 (1981)

Multienzyme reaction systems with simultaneous coenzyme regeneration have been investigated in a continuously operated membrane reactor at bench scale. NAD(H) covalently bound to polyethylene glycol with a molecular weight of 10(4) [PEG-10,000-NAD(H)] was used as coenzyme. It could be retained in the membrane reactor together with the enzymes. L-leucine dehydrogenase (LEUDH) was used as catalyst for the reductive amination of alpha-ketoisocaproate (2-oxo-4-methylpentanoic acid) to L-leucine. Formate dehydrogenase (FDH) was used for the regeneration of NADH. Kinetic experiments were carried out to obtain data which could be used in a kinetic model in order to predict the performance of an enzyme membrane reactor for the continuous production of L-leucine. The kinetic constants V(max) and k(m) of the enzymes are all in the same range regardless of whether native NAD(H) or PEG-10,000-NAD(H) is used as coenzyme. L-leucine was produced continuously out of alpha-ketoisocaproate for 48 days; a maximal conversion of 99.7% was reached. The space-time yield was 324 mmol/L day (or 42.5 g/L day).     

The occurrence of 4-methylthio-2-hydroxybutyrate in human urine

A method for determination of 4-methylthio-2-hydroxybutyrate and 4-methylthio-2-oxobutyrate in human urine has been devised, based on metoxime formation of the keto acid and a clean-up procedure using a strong anion-exchange resin AG 2 X 8 and ethyl acetate extraction. After alkylation, the compounds were quantified by GC, using a flame photometric sulfur-selective detector. A normal urinary excretion of 0.14 to 0.25 mmol/mol creatinine and 0.07 to 0.22 mmol/mol creatinine of the alpha-hydroxy and alpha-keto acid, respectively, was found, whereas a markedly elevated excretion of the hydroxy acid was noted in subjects with hypermethioninemia. The enzymatic reduction of 4-methylthio-2-oxobutyric acid by lactate dehydrogenase: NAD+ oxidoreductase (EC 1.1.1.17) was also studied. The Km and Kequil values for 4-methylthio-2-oxobutyrate were 1.41 mM and 0.92 X 10(8) M-1. The Vmax value of the enzyme at infinite concentrations of the two substrates was 7.2 mumol/s/mumol enzyme, which indicates low affinity and reduced catalytic activity compared to other known substrates of lactate dehydrogenase. The reaction product 4-methylthio-2-hydroxybutyrate was not inhibitory on the reaction. The M4 isoenzyme of lactate dehydrogenase (rabbit and pig muscle) possessed approximately 20% of the activity of the H4 isoenzyme (pig heart) for the substrate.     

Efficient production of (R)-(−)-2-hydroxy-4-phenylbutyric acid by recombinant Pichia pastoris expressing engineered d-lactate dehydrogenase from Lactobacillus plantarum with a single-site mutation

Bioprocess and Biosystems Engineering - (R)-2-hydroxy-4-phenylbutyric acid (R-HPBA) is a valuable intermediate for the synthesis of angiotensin-converting enzyme inhibitors. The asymmetric...     

A novel NADH-dependent carbonyl reductase from Kluyveromyces aestuarii and comparison of NADH-regeneration system for the synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate

To compare NADH-regeneration systems for the synthesis of (S)-4-chloro-3-hydroxybutanoate (ECHB), a novel NADH-dependent carbonyl reductase (KaCR1), which reduced ethyl 4-chloroacetoacetate (ECAA) to form (S)-ECHB, was screened and purified from Kluyveromyces aestuarii and a gene encoding KaCR1 was cloned. Glucose dehydrogenase (GDH) and formate dehydrogenase (FDH) were compared as enzymes for NADH regeneration using Escherichia coli cells coexpressing each enzyme with KaCR1. E. coli cells coexpressing GDH produced 45.6 g/l of (S)-ECHB from 50 g/l of ECAA and E. coli cells coexpressing FDH, alternatively, produced only 19.0 g/l. The low productivity in the case of FDH was suggested to result from the low activity and instability of FDH.     

Overproduction and nucleotide sequence of the respiratory D-lactate dehydrogenase of Escherichia coli.

Recombinant DNA plasmids containing the gene for the membrane-bound D-lactate dehydrogenase (D-LDH) of Escherichia coli linked to the promoter PL from lambda were constructed. After induction, the levels of D-LDH were elevated 300-fold over that of the wild type and amounted to 35% of the total cellular protein. The nucleotide sequence of the D-LDH gene was determined and shown to agree with the amino acid composition and the amino-terminal sequence of the purified enzyme. Removal of the amino-terminal formyl-Met from D-LDH was not inhibited in cells which contained these high levels of D-LDH.     

10-formyltetrahydrofolate dehydrogenase requires a 4'-phosphopantetheine prosthetic group for catalysis

10-Formyltetrahydrofolate dehydrogenase (FDH) consists of two independent catalytic domains, N- and C-terminal, connected by a 100-amino acid residue linker (intermediate domain). Our previous studies on structural organization and enzymatic properties of rat FDH suggest that the overall enzyme reaction, i.e. NADP(+)-dependent conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO(2), consists of two steps: (i) hydrolytic cleavage of the formyl group in the N-terminal catalytic domain, followed by (ii) NADP(+)-dependent oxidation of the formyl group to CO(2) in the C-terminal aldehyde dehydrogenase domain. In this mechanism, it was not clear how the formyl group is transferred between the two catalytic domains after the first step. This study demonstrates that the intermediate domain functions similarly to an acyl carrier protein. A 4'-phosphopantetheine swinging arm bound through a phosphoester bond to Ser(354) of the intermediate domain transfers the formyl group between the catalytic domains of FDH. Thus, our study defines the intermediate domain of FDH as a novel carrier protein and provides the previously lacking component of the FDH catalytic mechanism.     

Biotransformation of D-methionine into L-methionine in the cascade of four enzymes

D-Methionine was converted to L-methionine in a reaction system where four enzymes were used. D-amino acid oxidase (D-AAO) from Arthrobacter protophormiae was used for the complete conversion of D-methionine to 2-oxo-4-methylthiobutyric acid. Catalase was added to prevent 2-oxo-4-methylthiobutyric acid decarboxylation. In the second reaction step, L-phenylalanine dehydrogenase (L-PheDH) from Rhodococcus sp. was used to convert 2- oxo-4-methylthiobutyric acid to L-methionine, and formate dehydrogenase (FDH) from Candida boidinii was added for NADH regeneration. Enzyme kinetics of all enzymes was analyzed in detail. Mathematical models for separate reactions steps, as well as for the complete system were developed and validated in the batch reactor experiments. Complete conversion of D-methionine to L-methionine was achieved. Considering that both enzymes act on different substrates, such a system could be easily employed for the synthesis of other amino acids from D-isomer, as well as from the racemate of a certain amino acid (DL-amino acid).     

Continuous enzymatic transformation in an enzyme membrane reactor with simultaneous NAD(H) regeneration

Multienzyme reaction systems with simultaneous coenzyme regeneration have been investigated in a continuously operated membrane reactor at bench scale. NAD(H) covalently bound to polyethylene glycol with a molecular weight of 10⁴ [PEG-10,000-NAD(H)] was used as coenzyme. It could be retained in the membrane reactor together with the enzymes. L -leucine dehydrogenase (LEUDH) was used as catalyze for the reductive amination of α-ketoisocaproate (2-oxo-4-methylpentanoic acid) to L -leucine. Format dehydrogenease (FDH) was used for the regeneration of NADH. Kinetic experiments were carried out to obtain data which could be used in a kinetic model in order to predict the performance of an enzyme membrane reactor for the continuous production of L -leucine. The kinetic constants V_max and K_m of enzymes are all in the same range regardless of whether native NAD(H) or PEG-10,000-NAD(H) is used as coenzyme. L -leucine was produced continuously out of α-ketoisocaproate for 48 days; a maximal conversion of 99.7% was reached. The space-time yield was 324 mmol/L day (or 42.5 g/L day).     

Enantioselective Metabolism of Chiral 3-Phenylbutyric Acid, an Intermediate of Linear Alkylbenzene Degradation, by Rhodococcus rhodochrous PB1

Rhodococcus rhodochrous PB1 was isolated from compost soil by selective culture with racemic 3-phenylbutyric acid as the sole carbon and energy source. Growth experiments with the single pure enantiomers as well as with the racemate showed that only one of the two enantiomers, (R)-3-phenylbutyric acid, supported growth of strain PB1. Nevertheless, (S)-3-phenylbutyric acid was cometabolically transformed to, presumably, (S)-3-(2,3-dihydroxyphenyl)butyric acid (the absolute configuration at the C-3 atom is not known yet) by (R)-3-phenylbutyric acid-grown cells of strain PB1, as shown by (sup1)H nuclear magnetic resonance spectroscopy of the partially purified compound and gas chromatography-mass spectrometry analysis of the trimethylsilyl derivative. Oxygen uptake rates suggest that either 3-phenylpropionic acid or cinnamic acid (trans-3-phenyl-2-propenoic acid) is the substrate for aromatic ring hydroxylation. This view is substantiated by the fact that 3-(2,3-dihydroxyphenyl)propionic acid was a substrate for meta cleavage in cell extracts of (R)-3-phenylbutyric acid-grown cells of strain PB1. Gas chromatography-mass spectrometry analysis of trimethylsilane-treated ethyl acetate extracts of incubation mixtures showed that both the meta-cleavage product, 2-hydroxy-6-oxo-2,4-nonadiene-1,9-dicarboxylic acid, and succinate, a hydrolysis product thereof, were formed during such incubations.     

Characterization of d-lactate dehydrogenase from Pediococcus acidilactici that converts phenylpyruvic acid into phenyllactic acid

The gene coding for D-lactate dehydrogenase (D-LDH) from Pediococcus acidilactici DSM 20284 was cloned and expressed in E. coli. The recombinant enzyme was purified by nickel-affinity chromatography. It converted phenylpyruvic acid (PPA) to 3-phenyllactic acid maximally at 30°C and pH 5.5 with a specific activity of 140 and 422 U/mg for PPA and pyruvate, respectively. The K(m), turnover number (k(cat)), and catalytic efficiency (k(cat)/K(m)) for PPA were 2.9 mM, 305 s(-1), and 105 mM(-1) s(-1), respectively.     

A novel reactor concept for the enzymatic reduction of poorly soluble ketones

Reductions of poorly soluble ketones often suffer from low total turnover numbers conferring to the coenzyme and large volumes which are needed for the conversion. The novel emulsion membrane reactor overcomes these limitations. From an emulsion consisting of an organic substrate and an aqueous buffer phase, the aqueous phase is separated selectively by using a hydrophilic ultrafiltration membrane and fed to a subsequent enzyme membrane reactor. The product outflow is recirculated to the emulsion stirred vessel and, due to the partition coefficients, the aqueous phase is recharged with substrate while the product is extracted. This new reactor concept will be compared to the classical enzyme membrane reactor. The latter was operated under the same conditions over a period of 4 months at a space-time yield of 21.2 g l⁻¹ day⁻¹. As a model system the enantioselective reduction of 2-octanone to (S)-2-octanol (ee > 99.5%) is used, carried out by a carbonyl reductase from Candida parapsilosis. NADH is regenerated by formate dehydrogenase from Candida boidinii. In comparison to the classical enzyme membrane reactor the total turnover number could be increased by a factor 9 using the novel emulsion membrane reactor.     

Biomimetic-dye affinity adsorbents for enzyme purification: application to the one-step purification of Candida boidinii formate dehydrogenase

Formate dehydrogenase (FDH, EC 1.2.1.2) was purified from Candida boidinii cells in a single step by biomimetic-dye affinity chromatography. For this purpose, seven' biomimetic analogues of the monochlorotriazine dye, Cibacron(R) Blue 3GA (CB3GA), and parent dichloro-triazine dye, Vilmafix((R)) Blue A-R (VBAR), bearing a car-boxylated structure as their terminal biomimetic moiety, were immobilized on crosslinked agarose gel, Ultrogel((R)) A6R. The corresponding new biomimetic-dye adsorbents, along with nonbiomimetic adsorbents bearing CB3GA and VBAR, were evaluated for their ability to purify FDH from extracts obtained after press-disintegration of C. boidinii cells. Optimal conditions for maximizing specific activity of FDH in starting extracts (1.8 U/mg) were realized when cell growth was performed on 4% methanol, and press disintegration proceeded in four consecutive passages before the homogenate was left to stand for 1 h (4 degrees C). When compared to nonbiomimetic adsorbents, biomimetic adsorbents exhibited higher purifying ability. Furthermore, one immobilized biomimetic dye, bearing as its terminal biomimetic moiety mercap-topyruvic acid linked on the chlorotriazine ring (BM6), displayed the highest purifying ability. Adsorption equilibrium data which were obtained for the BM6 adsorbent in a batch system corresponded well to the Langmuir isotherm and, in addition, breakthrough curves were taken for protein and FDH adsorption in a fixed bed of BM6 adsorbent. The dissociation constant ( K(D)) of the complex between immobilized BM6 and FDH was found to equal 0.05 muM. Adsorbent BM6 was employed in the purification of FDH from a 18-L culture of C. boidinii in a single step (60% overall yield of FDH). The purified FDH afforded a single-band on sodium dodecyl sulphate poly-acrylamide gel electrophoresis, and a specific activity of 7,0 U/mg (30 degrees C). (c) 1995 John Wiley & Sons, Inc.     

A 19F-NMR study of the membrane-binding region of D-lactate dehydrogenase of Escherichia coli.

D-Lactate dehydrogenase (D-LDH) is a membrane-associated respiratory enzyme of Escherichia coli. The protein is composed of 571 amino acid residues with a flavin adenine dinucleotide (FAD) cofactor, has a molecular weight of approximately 65,000, and requires lipids or detergents for full activity. We used NMR spectroscopy to investigate the structure of D-LDH and its interaction with phospholipids. We incorporated 5-fluorotryptophan (5F-Trp) into the native enzyme, which contains five tryptophan residues, and into mutant enzymes, where a sixth tryptophan is substituted into a specific site by oligonucleotide-directed mutagenesis, and studied the 5F-Trp-labeled enzymes using 19F-NMR spectroscopy. In this way, information was obtained about the local environment at each native and substituted tryptophan site. Using a nitroxide spin-labeled fatty acid, which broadens the resonance from any residue within 15 A, we have established that the membrane-binding area of the protein includes the region between Tyr 228 and Phe 369, but is not continuous within this region. This conclusion is strengthened by the results of 19F-NMR spectroscopy of wild-type enzyme labeled with fluorotyrosine or fluorophenylalanine in the presence and absence of a nitroxide spin-labeled fatty acid. These experiments indicate that 9-10 Phe and 3-4 Tyr residues are located near the lipid phase.     

Characterization of D-lactate dehydrogenase producing D-3-phenyllactic acid from Pediococcus pentosaceus

D-Lactate dehydrogenase (D-LDH) from Pediococcus pentosaceus ATCC 25745 was found to produce D-3-phenyllactic acid from phenylpyruvate. The optimum pH and temperature for enzyme activity were pH 5.5 and 45 °C. The Michaelis-Menten constant (K(m)), turnover number (k(cat)), and catalytic efficiency (k(cat)/K(m)) values for the substrate phenylpyruvate were estimated to be 1.73 mmol/L, 173 s(-1), and 100 (mmol/L)(-1) s(-1) respectively.     

FDH: an aldehyde dehydrogenase fusion enzyme in folate metabolism

FDH (10-formyltetrahydrofolate dehydrogenase, Aldh1L1, EC 1.5.1.6) converts 10-formyltetrahydrofolate (10-formyl-THF) to tetrahydrofolate and CO(2) in a NADP(+)-dependent reaction. It is a tetramer of four identical 902 amino acid residue subunits. The protein subunit is a product of a natural fusion of three unrelated genes and consists of three distinct domains. The N-terminal domain of FDH (residues 1-310) carries the folate binding site and shares sequence homology and structural topology with other enzymes utilizing 10-formyl-THF as a substrate. In vitro it functions as 10-formyl-THF hydrolase, and evidence indicate that this activity is a part of the overall FDH mechanism. The C-terminal domain of FDH (residues 400-902) originated from an aldehyde dehydrogenase-related gene and is capable of oxidation of short-chain aldehydes to corresponding acids. Similar to classes 1 and 2 aldehyde dehydrogenases, this domain exists as a tetramer and defines the oligomeric structure of the full-length enzyme. The two catalytic domains are connected by an intermediate linker (residues 311-399), which is a structural and functional homolog of carrier proteins possessing a 4'-phosphopantetheine prosthetic group. In the FDH mechanism, the intermediate linker domain transfers a formyl, covalently attached to the sulfhydryl group of the phosphopantetheine arm, from the N-terminal domain to the C-terminal domain. The overall FDH mechanism is a coupling of two sequential reactions, a hydrolase and a formyl dehydrogenase, bridged by a substrate transfer step. In this mechanism, one domain provides the folate binding site and a hydrolase catalytic center to remove the formyl group from the folate substrate, another provides a transfer vehicle between catalytic centers and the third one contributes the dehydrogenase machinery further oxidizing formyl to CO(2).     

A highly active, soluble mutant of the membrane-associated (S)-mandelate dehydrogenase from Pseudomonas putida.

(S)-Mandelate dehydrogenase (MDH) from Pseudomonas putida, a member of the flavin mononucleotide-dependent alpha-hydroxy acid oxidase/dehydrogenase family, is a membrane-associated protein, in contrast to the more well-characterized members of this protein family including glycolate oxidase (GOX) from spinach. In a previous study [Mitra, B., et al. (1993) Biochemistry 32, 12959-12967], the membrane association of MDH was correlated to a 53 amino acid segment in the interior of the primary sequence by construction of a chimeric enzyme, MDH-GOX1, in which the membrane-binding segment in MDH was deleted and replaced with the corresponding 34 amino acid segment from the soluble GOX. Though MDH-GOX1 was soluble, it was an inefficient, nonspecific enzyme that involved a different transition state for the catalyzed reaction from that of the wild-type MDH. In the present study, it is shown that the membrane-binding segment in MDH is somewhat shorter, approximately 39 residues long. Partial or total deletion of this segment disrupts membrane localization of MDH. This segment is not important for substrate oxidation activity. A new chimera, MDH-GOX2, was created by replacing this shorter membrane-binding segment from MDH with the corresponding 20 amino acid segment from GOX. The soluble MDH-GOX2 is very similar to the wild-type membrane-bound enzyme in its spectroscopic properties, substrate specificity, catalytic activity, kinetic mechanism, and lack of reactivity toward oxygen. Therefore, it should prove to be a highly useful model for structural studies of MDH.