Modeling substrate binding in Thermus thermophilus isopropylmalate dehydrogenase.

The Thermus thermophilus 3-isopropylmalate dehydrogenase (IPMDH) and Escherichia coli isocitrate dehydrogenase (ICDH) are two functionally and evolutionarily related enzymes with distinct substrate specificities. To understand the determinants of substrate specificities of the two proteins, the substrate and coenzyme in IPMDH were docked into their respective binding sites based on the published structure for apo IPMDH and its sequence and structural homology to ICDH. This modeling study suggests that (1) the substrate and coenzyme (NAD) binding modes of IPMDH are significantly different from those of ICDH, (2) the interactions between the substrates and coenzymes help explain the differences in substrate specificities of IPMDH and ICDH, and (3) binding of the substrate and coenzyme should induce a conformational change in the structure of IPMDH.     

Coenzyme A- and NADH-dependent esterase activity of methylmalonate semialdehyde dehydrogenase.

Methylmalonate semialdehyde dehydrogenase purified to homogeneity from rat liver possesses, in addition to its coupled aldehyde dehydrogenase and CoA ester synthetic activity, the ability to hydrolyze p-nitrophenyl acetate. The following observations suggest that this activity is an active site phenomenon: (a) p-nitrophenyl acetate hydrolysis was inhibited by malonate semialdehyde, substrate for the dehydrogenase reaction; (b) p-nitrophenyl acetate was a strong competitive inhibitor of the dehydrogenase activity; (c) NAD+ and NADH activated the esterase activity; (d) coenzyme A, acceptor of acyl groups in the dehydrogenase reaction, accelerated the esterase activity; and (e) the product of the esterase reaction proceeding in the presence of coenzyme A was acetyl-CoA. These findings suggest that an S-acyl enzyme (thioester intermediate) is likely common to both the esterase reaction and the aldehyde dehydrogenase/CoA ester synthetic reaction.     

Inducibility of liver cytosolic aldehyde dehydrogenase activity in various animal species

1. The inducibility of hepatic cytosolic aldehyde dehydrogenase activity was studied in rat, mouse, guinea pig, chicken, frog, salamander and rainbow trout, by using two different types of inducers of drug metabolism. 2. Phenobarbital (a type I inducer of drug metabolizing enzymes) increased total liver cytosolic aldehyde dehydrogenase activity (up to 20-fold) in a genetically defined substrain of responsive rats (RR) and only slightly, if at all, in a non-responsive substrain (rr). On the contrary, both types of rats showed a highly induced aldehyde dehydrogenase activity after treatment with methylcholanthrene (a type II inducer). Phenobarbital is affecting mainly an isozyme of aldehyde dehydrogenase which is best measured with propionaldehyde as the substrate and NAD as the coenzyme (P/NAD). 3. Administration of phenobarbital to mice produced only a slight increase (2-fold) in the P/NAD aldehyde dehydrogenase activity. 4. Methylcholanthrene treatment caused a 2-fold increase of the hepatic P/NAD aldehyde dehydrogenase activity in the chicken. 5. In the guinea pig, phenobarbital produced an approximate 3-fold increase of the P/NAD activity. Methylcholanthrene had a similar effect, although to a lesser extent. 6. In the salamander, a 4-fold increase was detected in the enzyme activity measured with benzaldehyde as the substrate and NADP as the coenzyme (B/NADP), after treatment with either phenobarbital or methylcholanthrene. 7. The hepatic aldehyde dehydrogenase activities were found unchanged in the rainbow trout, after treatment with phenobarbital or 2,3,7,8-tetrachlorodibenzo-p-dioxin. 8. The rat model remains the only one examined that shares with human hepatocytes strong inducibility of the B/NADP aldehyde dehydrogenase isozyme upon treatment with polycyclic aromatic hydrocarbons.     

谷氨酸脱氢酶与醇脱氢酶的共表达及其在制备(R)-2-氯-1-苯乙醇中的应用

【背景】醇脱氢酶AdhS能催化不对称还原反应制备(R)-2-氯-1-苯乙醇,但由于自身再生辅酶NADH的能力不足,需要辅酶再生酶协助其再生NADH。谷氨酸脱氢酶能以谷氨酸为底物,再生辅酶NAD(P)H,具有辅酶再生酶的潜力。【目的】克隆表达谷氨酸脱氢酶基因gdhA,构建谷氨酸脱氢酶GdhA与醇脱氢酶AdhS的大肠杆菌共表达体系,提高AdhS制备(R)-2-氯-1-苯乙醇的转化效率。【方法】从枯草芽孢杆菌(Bacillus subtilis) 168中克隆基因gdhA,并在大肠杆菌(Escherichia coli) BL21(DE3)中表达,分析辅酶再生活力;再与醇脱氢酶AdhS共表达,优化表达条件;分析不同辅酶再生方案对制备(R)-2-氯-1-苯乙醇的转化效率的影响。【结果】谷氨酸脱氢酶GdhA再生NADH的比活力为694 U/g。经GdhA与AdhS的共表达及表达条件优化后,制备(R)-2-氯-1-苯乙醇的转化效率达465 U/L。经比较,GdhA协助再生辅酶NADH,可使AdhS制备(R)-2-氯-1-苯乙醇的转化效率提高到约3倍。【结论】谷氨酸脱氢酶GdhA为NADH高效再生酶,与醇脱氢酶AdhS共表达可显著提高AdhS制备(R)-2-氯-1-苯乙醇的转化效率。     

Covalent binding of an NAD analogue to liver alcohol dehydrogenase resulting in an enzyme-coenzyme complex not requiring exogenous coenzyme for activity.

1. The NAD analogue, N6-[N-(6-aminohexyl)carbamoylmethyl]-NAD, was covalently bound to horse liver alcohol dehydrogenase in a carbodiimide-mediated reaction and in such a way that it was active with the very same enzyme molecule to which it was coupled. 2. The degree of substitution, i.e. the number of NAD analogues per enzyme subunit, could be varied (0.3-1.6). In one preparation 1.6 coenzyme molecules were bound per subunit; the alcohol dehydrogenase activity of this preparation was 40% of the activity obtained after addition of free NAD in excess. 3. It was calculated that every fourth active site of this preparation was provided with a covalently bound functioning coenzyme analogue, and that this analogue had a cycling rate of about 40 000 cycles/h in a coupled substrate assay. 4. The presence of the covalently bound coenzyme made the active sites difficult to inhibit with a competitive inhibitor. For example, 10 mM AMP inhibited the activity of the preparation by 50% whereas a reference system containing native alcohol dehydrogenase was inhibited by 80% in spite of the fact that the reference system contained about 20 000 times as high a concentration of coenzyme.     

Kinetic studies of dogfish liver glutamate dehydrogenase.

Initial-rate studies were made of the oxidation of L-glutamate by NAD+ and NADP+ catalysed by highly purified preparations of dogfish liver glutamate dehydrogenase. With NAD+ as coenzyme the kinetics show the same features of coenzyme activation as seen with the bovine liver enzyme [Engel & Dalziel (1969) Biochem. J. 115, 621--631]. With NADP+ as coenzyme, initial rates are much slower than with NAD+, and Lineweaver--Burk plots are linear over extended ranges of substrate and coenzyme concentration. Stopped-flow studies with NADP+ as coenzyme give no evidence for the accumulation of significant concentrations of NADPH-containing complexes with the enzyme in the steady state. Protection studies against inactivation by pyridoxal 5'-phosphate indicate that NAD+ and NADP+ give the same degree of protection in the presence of sodium glutarate. The results are used to deduce information about the mechanism of glutamate oxidation by the enzyme. Initial-rate studies of the reductive amination of 2-oxoglutarate by NADH and NADPH catalysed by dogfish liver glutamate dehydrogenase showed that the kinetic features of the reaction are very similar with both coenzymes, but reactions with NADH are much faster. The data show that a number of possible mechanisms for the reaction may be discarded, including the compulsory mechanism (previously proposed for the enzyme) in which the sequence of binding is NAD(P)H, NH4+ and 2-oxoglutarate. The kinetic data suggest either a rapid-equilibrium random mechanism or the compulsory mechanism with the binding sequence NH4+, NAD(P)H, 2-oxoglutarate. However, binding studies and protection studies indicate that coenzyme and 2-oxoglutarate do bind to the free enzyme.     

Induction of aldehyde dehydrogenase by polycyclic aromatic hydrocarbons in rats

Short-term intragastric administration of selected polycyclic aromatic hydrocarbons (100 mg/kg daily for 4 days) to male Wistar rats resulted in marked changes in liver cytosolic aldehyde dehydrogenase activity. Non-carcinogenic anthracene, phenanthrene and chrysene produced a 2.5–3-fold increase in the activity assayed with propionaldehyde as substrate and NAD as coenzyme. Weakly carcinogenic 1,2-benzanthracene enhanced aldehyde dehydrogenase activity 9-fold and the potent carcinogens 3,4-benzpyrene and 3-methylcholanthrene 30-fold. With benzaldehyde as substrate and NADP as coenzyme the differences between the groups were even more pronounced. Somewhat similar but less manifest effects on aldehyde dehydrogenase activity were detected also in the liver microsomes and in the postmitochondrial fractions of the small intestinal mucosa. On the basis of their ability to induce aldehyde dehydrogenase activity the compounds could be divided into three groups. This classification was found to correlate well with the carcinogenic potency of the compounds. It appeared that the exposure to polycyclic aromatic hydrocarbons, especially the carcinogenic ones, was followed by synthesis of a new aldehyde dehydrogenase form. This new form was differentiated from the normally existing cytosolic aldehyde dehydrogenase by its ability to oxidize benzaldehyde in the presence of NADP.     

The importance of arginine residues in the catalytic and regulatory functions of bovine-liver glutamate dehydrogenase.

Bovine liver glutamate dehydrogenase reacts rapidly with 2,3-butanedione to yield modified enzyme with 29% of its original maximum activity, but no change in its Michaelis constants for substrates and coenzymes. No significant reduction in the inactivation rate is produced by the addition of the allosteric activator ADP or inhibitor GTP, while partial protection against inactivation is provided by the coenzyme NAD+ or substrate 2-oxoglutarate when added separately. The most marked decrease in the rate of inactivation (about 10-fold) is provided by the combined addition of NAD+ and 2-oxoglutarate, suggesting that modification takes place in the region of the active site. Reaction with 2,3-butanedione also results in loss of the ability of the enzyme to be activated by ADP. Addition of ADP (but not NAD+, 2-oxoglutarate or GTP) to the incubation mixture protects markedly against the loss of activatability of ADP. It is concluded that 2,3-butanedione produces two distinguishable effects on glutamate dehydrogenase: a relatively specific modification of the regulatory ADP site and a distinct modification in the active center. Reaction of two arginyl residues per peptide chain appears to be responsible for disruption of the ADP activation property of the enzyme, while alteration of a maximum of five arginyl residues can be related to the reduction of maximum catalytic activity. Electrostatic interactions between the positively charged arginine groups and the negatively charged substrate, coenzyme and allosteric purine nucleotide may be important for the normal function of glutamate dehydrogenase.     

甲酸脱氢酶及其在手性生物制造中的应用

氧化还原生物合成体系在绿色生物制造手性化合物中具有重要应用价值.甲酸脱氢酶(formate dehydrogenase,FDH)能氧化甲酸盐生成二氧化碳,同时将NAD(P)+还原为NAD(P)H,是氧化还原生物合成中辅酶再生体系的关键酶.但天然的FDH催化效率低、稳定性差、辅酶利用率不高等缺点制约了其在工业生产中的应用...     

NAD-dependent, PQQ-containing methanol dehydrogenase: a bacterial dehydrogenase in a multienzyme complex

Cell-free extracts of methanol-grown Nocardia sp. 239 only show significant dye-linked methanol-oxidizing activity when NAD+ is added to the assay mixture. This activity resides in a multienzyme complex which could be resolved into 3 components, namely the methanol dehydrogenase, NAD-dependent aldehyde dehydrogenase and NADH dehydrogenase. In its dissociated form, the methanol dehydrogenase no longer shows dye reduction and although rises in the absorbance values around 340 nm are seen on addition of methanol plus NAD+ to the enzyme, this is not due to NADH production. However, dye reduction (NAD dependent) could be restored on incubating methanol dehydrogenase with the corresponding NADH dehydrogenase, obtained from the enzyme complex. It is concluded that this novel methanol dehydrogenase transfers the reducing equivalents, derived from methanol, directly to its associated NADH dehydrogenase via a mechanism in which NAD+ and PQQ are involved.     

Multiple forms and specificity of coniferyl alcohol dehydrogenase from cambial regions of higher plants

Alcohol dehydrogenases of 89 species of plants, from the Bryophyta, Pteridophyta, Gymnosperms and Angiosperms were examined by starch gel electrophoresis for their substrate and coenzyme specificities. High activities and multiple bands were observed with EtOH and NAD in most species. The same, but weaker banding patterns were also observed with benzyl alcohol and salicin. When coniferyl alcohol was used as substrate, activity was found only with NADP as coenzyme and the resulting bands were distinct from those obtained with the other substrates. Most plants tested had only one or occasionally a second coniferyl alcohol dehydrogenase band. Salix species were an exception, with multiple bands found in each of the species tested.     

Regulation of ox liver glutamate dehydrogenase activity by coenzymes

The effects of coenzymes NAD(P) and NAD(P)H on the kinetics of the ox liver glutamate dehydrogenase reaction have been studied. The oxidized coenzymes were shown to activate alpha-ketoglutarate amination at inhibiting concentrations of NADH and NADPH. The reduced coenzymes, NADH and NADPH, inhibit glutamate deamination with both NAD and NADP as coenzymes. The data obtained are discussed in terms of literature data on the mechanisms of the coenzyme effects on the glutamate dehydrogenase activity and are inconsistent with the theory of direct ligand--ligand interactions. It was shown that the peculiarities of the glutamate dehydrogenase kinetics can easily be interpreted in the light of the two state models.     

Succinic semialdehyde dehydrogenase of wheat grain

Succinic semialdehyde dehydrogenase (EC 1.2.1.16) was purified 74-fold from wheat grain (Triticum durum Desf.). The enzyme appears quite specific for succinic semialdehyde (SSA). Both NAD and NADP support the oxidation of the substrate, but the former is 7-fold more active than the latter. The optimum pH for activity is around 9; the enzyme is stable in the pH range 6–9 and retains its whole activity up to 40°C. The enzyme activity is strongly dependent on the presence of mercaptoethanol, other thiol compounds being much less effective. Kinetic data support the formation of a ternary complex between enzyme, substrate and coenzyme. The K _m for SSA and for NAD are 7.4x10^-6 M and 2x10^-4 M, respectively. The molecular weight of the enzyme protein was estimated by gel-filtration to be about 130,000.Abbreviations GABA -aminobutyric acid - GABA-T -aminobutyric acid transaminase - ME mercaptoethanol - SSA succinic semialdehyde - SSA-DH succinic semialdehyde dehydrogenase     

Enhancement of coenzyme binding by a single point mutation at the coenzyme binding domain of E. coli lactaldehyde dehydrogenase

Phenylacetaldehyde dehydrogenase (PAD) and lactaldehyde dehydrogenase (ALD) share some structural and kinetic properties. One difference is that PAD can use NAD+ and NADP+, whereas ALD only uses NAD+. An acidic residue has been involved in the exclusion of NADP+ from the active site in pyridine nucleotide-dependent dehydrogenases. However, other factors may participate in NADP+ exclusion. In the present work, analysis of the sequence of the region involved in coenzyme binding showed that residue F180 of ALD might participate in coenzyme specificity. Interestingly, F180T mutation rendered an enzyme (ALD-F180T) with the ability to use NADP+. This enzyme showed an activity of 0.87 micromol/(min * mg) and K(m) for NADP+ of 78 microM. Furthermore, ALD-F180T exhibited a 16-fold increase in the V(m) /K(m) ratio with NAD+ as the coenzyme, from 12.8 to 211. This increase in catalytic efficiency was due to a diminution in K(m) for NAD+ from 47 to 7 microM and a higher V(m) from 0.51 to 1.48 micromol/(min * mg). In addition, an increased K(d) for NADH from 175 (wild-type) to 460 microM (mutant) indicates a faster product release and possibly a change in the rate-limiting step. For wild-type ALD it is described that the rate-limiting step is shared between deacylation and coenzyme dissociation. In contrast, in the present report the rate-limiting step in ALD-F180T was determined to be exclusively deacylation. In conclusion, residue F180 participates in the exclusion of NADP+ from the coenzyme binding site and disturbs the binding of NAD+.     

荧光假单胞菌短链脱氢酶的克隆、表达及酶学性质分析

为了研究荧光假单胞菌中短链脱氢酶的生理角色和催化特性,从荧光假单胞菌Pseudomonas fluorescens GIM1.49基因组DNA克隆表达了一个短链脱氢酶的编码基因pfd,并分析了该基因产物的酶学性质。基因pfd全长684 bp,编码227个氨基酸,推算分子量为24.2 kDa。将携带短链脱氢酶基因的重组质粒pET28b-pfd转入大肠杆菌BL21(DE3) 进行表达,得到了28 kDa的表达产物。重组荧光假单胞菌短链脱氢酶 (PFD) 能氧化4-氯-3-羟基丁酸乙酯、1-苯乙醇、苯甲醇、仲丁醇和还原4-氯-乙酰乙酸乙酯、2-溴-苯乙酮、4-溴-苯乙酮等底物。以4-氯-3-羟基丁酸乙酯为底物时活力最高,Km值为186.90 mmol/L,Vmax为89.56 U/mg。氧化4-氯-3-羟基丁酸乙酯时,最适反应温度和pH分别为12 ℃和10.5,倾向于利用NAD+作辅酶;而还原4-氯-乙酰乙酸乙酯时,最适温度和pH为24 ℃和8.8,倾向于利用NADPH作辅酶。重组PFD能耐受50％ (V/V) 的甲醇等有机助溶剂,Ca2+ (1 mmol/L) 和EDTA (5 mmol/L) 对其酶活有一定的促进作用。上述结果表明,重组PFD是一个新型的短链脱氢酶,其代谢角色推测与卤代次级醇的氧化降解有关。     

The ternary complex of Pseudomonas aeruginosa alcohol dehydrogenase with NADH and ethylene glycol

Pseudomonas aeruginosa alcohol dehydrogenase (PaADH; ADH, EC 1.1.1.1) catalyzes the reversible oxidation of primary and secondary alcohols to the corresponding aldehydes and ketones, using NAD as coenzyme. We crystallized the ternary complex of PaADH with its coenzyme and a substrate molecule and determined its structure at a resolution of 2.3 A, using the molecular replacement method. The PaADH tetramer comprises four identical chains of 342 amino acid residues each and obeys ~222-point symmetry. The PaADH monomer is structurally similar to alcohol dehydrogenase monomers from vertebrates, archaea, and bacteria. The stabilization of the ternary complex of PaADH, the coenzyme, and the poor substrate ethylene glycol (k(cat) = 4.5 sec(-1); Km > 200 mM) was due to the blocked exit of the coenzyme in the crystalline state, combined with a high (2.5 M) concentration of the substrate. The structure of the ternary complex presents the precise geometry of the Zn coordination complex, the proton-shuttling system, and the hydride transfer path. The ternary complex structure also suggests that the low efficiency of ethylene glycol as a substrate results from the presence of a second hydroxyl group in this molecule.     

Structural evidence for ligand-induced sequential conformational changes in glyceraldehyde 3-phosphate dehydrogenase

Glyceraldehyde 3-phosphate dehydrogenase is a tetramer of four chemically identical subunits which requires the cofactor nicotinamide adenine dinucleotide (NAD) for activity. The structure of the holo-enzyme from Bacillus stearothermophilus has recently been refined using X-ray data to 2.4 A resolution. This has facilitated the structure determination of both the apo-enzyme and the enzyme with one molecule of NAD bound to the tetramer. These structures have been refined at 4 A resolution using the constrained-restrained parameter structure factor least-squares refinement program CORELS. When combined with individual atomic temperature factors from the holo-enzyme, these refined models give crystallographic R factors of 30.2% and 30.4%, respectively, for data to 3 A resolution. The apo-enzyme has 222 molecular symmetry, and the subunit structure is related to that of the holo-enzyme by an approximate rigid-body rotation of the coenzyme binding domain by 4.3 degrees with respect to the catalytic domains, which form the core of the tetramer. The effect of this rotation is to shield the coenzyme and active site from solvent in the holo-enzyme. In addition to the rigid-body rotation, there is a rearrangement of several residues involved in NAD binding. The structure of the 1 NAD enzyme is asymmetric. The subunit which contains the bound NAD adopts a conformation very similar to that of a holo-enzyme subunit, while the other three unliganded subunits are very similar to the apo-enzyme conformation. This result provides unambiguous evidence for ligand-induced sequential conformational changes in B. stearothermophilus glyceraldehyde 3-phosphate dehydrogenase.     

Molecular,biochemical, and functional characterization of a Nudix hydrolase protein that stimulates the activity of a nicotinoprotein alcohol dehydrogenase

The cytoplasmic coenzyme NAD(+)-dependent alcohol (methanol) dehydrogenase (MDH) employed by Bacillus methanolicus during growth on C(1)-C(4) primary alcohols is a decameric protein with 1 Zn(2+)-ion and 1-2 Mg(2+)-ions plus a tightly bound NAD(H) cofactor per subunit (a nicotinoprotein). Mg(2+)-ions are essential for binding of NAD(H) cofactor in MDH protein expressed in Escherichia coli. The low coenzyme NAD(+)-dependent activity of MDH with C(1)-C(4) primary alcohols is strongly stimulated by a second B. methanolicus protein (ACT), provided that MDH contains NAD(H) cofactor and Mg(2+)-ions are present in the assay mixture. Characterization of the act gene revealed the presence of the highly conserved amino acid sequence motif typical of Nudix hydrolase proteins in the deduced ACT amino acid sequence. The act gene was successfully expressed in E. coli allowing purification and characterization of active ACT protein. MDH activation by ACT involved hydrolytic removal of the nicotinamide mononucleotide NMN(H) moiety of the NAD(H) cofactor of MDH, changing its Ping-Pong type of reaction mechanism into a ternary complex reaction mechanism. Increased cellular NADH/NAD(+) ratios may reduce the ACT-mediated activation of MDH, thus preventing accumulation of toxic aldehydes. This represents a novel mechanism for alcohol dehydrogenase activity regulation.     

Covalent binding of an NAD+ analogue to horse liver alcohol dehydrogenase in a ternary complex with pyrazole

Examination of the model of the fixation site of the adenosine phosphate part of NAD+ on horse liver alcohol dehydrogenase led us to synthesize a NAD+ analogue N6-[N-(8-amino-3,6-dioxaoctyl)carbamoylmethyl]-NAD+ in order to alkylate the carboxylic acid group of Asp-273 and to convert the normally dissociable coenzyme into a permanently bound prosthetic group. This NAD+ analogue is coupled to the horse liver alcohol dehydrogenase in the ternary complex formed with pyrazole. In these conditions the degree of fixation varies between 0.4 and 0.58 coenzyme molecule/enzyme subunit molecule. The N6-[N-(8-amino-3,6-dioxaoctyl)carbamoylmethyl]NAD+ acts as a true prosthetic group which can be reduced and reoxidized by a coupled substrate reaction and the internal activity of this holoenzyme corresponds to the amount of analogue incorporated.     

Cofactor engineering of Lactobacillus brevis alcohol dehydrogenase by computational design

The R‐specific alcohol dehydrogenase from Lactobacillus brevis (Lb‐ADH) catalyzes the enantioselective reduction of prochiral ketones to the corresponding secondary alcohols. It is stable and has broad substrate specificity. These features make this enzyme an attractive candidate for biotechnological applications. A drawback is its preference for NADP(H) as a cofactor, which is more expensive and labile than NAD(H). Structure‐based computational protein engineering was used to predict mutations to alter the cofactor specificity of Lb‐ADH. Mutations were introduced into Lb‐ADH and tested against the substrate acetophenone, with either NAD(H) or NADP(H) as cofactor. The mutant Arg38Pro showed fourfold increased activity with acetophenone and NAD(H) relative to the wild type. Both Arg38Pro and wild type exhibit a pH optimum of 5.5 with NAD(H) as cofactor, significantly more acidic than with NADP(H). These and related Lb‐ADH mutants may prove useful for the green synthesis of pharmaceutical precursors.