Simultaneous analysis of NAD- and NADP-linked activities of dual nucleotide-specific dehydrogenases. Application to Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase.

Levy, Haim; Daouk, Ghaleb H.

doi:10.1016/s0021-9258(17)30089-3

Cited by 26 publications

(6 citation statements)

References 16 publications

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“…The K m for NADP + for R46A G6PD is so high (6) that it is impractical to test the NADP-linked reaction for NADPH inhibition. As the thionicotinamide analogues, S-NADP + and S-NAD + , have substantially lower K m values than the corresponding natural coenzymes, while the kinetic mechanisms for the reactions in which they participate remain unaltered (20), we examined NADPH inhibition with respect to S-NADP + with R46A G6PD and found that it is noncompetitive. Likewise, both S-NADH and NADH inhibit noncompetitively with respect to NAD + , consistent with the NAD-linked reaction mechanism remaining random.…”

Section: Resultsmentioning

confidence: 99%

“…This unusual dual coenzyme specificity is accompanied by two other catalytic features: higher k cat and coenzyme K m values for the NAD-linked than the NADPlinked reaction (10) and different kinetic mechanisms, ordered for the NADP-linked and steady-state random for the NAD-linked reactions (18). This difference in kinetic mechanisms is the basis for an unusual regulatory feature of the enzyme, whereby NADPH and G6P can regulate the enzyme's selection of coenzyme utilization (20,21). The present investigation provides some insights into these characteristics of the enzyme.…”

Section: Discussionmentioning

confidence: 99%

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Delineation of the Roles of Amino Acids Involved in the Catalytic Functions of Leuconostoc mesenteroides Glucose 6-Phosphate Dehydrogenase

et al. 2000

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The roles of particular amino acids in substrate and coenzyme binding and catalysis of glucose-6-phosphate dehydrogenase of Leuconostoc mesenteroides have been investigated by site-directed mutagenesis, kinetic analysis, and determination of binding constants. The enzyme from this species has functional dual NADP(+)/NAD(+) specificity. Previous investigations in our laboratories determined the three-dimensional structure. Kinetic studies showed an ordered mechanism for the NADP-linked reaction while the NAD-linked reaction is random. His-240 was identified as the catalytic base, and Arg-46 was identified as important for NADP(+) but not NAD(+) binding. Mutations have been selected on the basis of the three-dimensional structure. Kinetic studies of 14 mutant enzymes are reported and kinetic mechanisms are reported for 5 mutant enzymes. Fourteen substrate or coenzyme dissociation constants have been measured for 11 mutant enzymes. Roles of particular residues are inferred from k(cat), K(m), k(cat)/K(m), K(d), and changes in kinetic mechanism. Results for enzymes K182R, K182Q, K343R, and K343Q establish Lys-182 and Lys-343 as important in binding substrate both to free enzyme and during catalysis. Studies of mutant enzymes Y415F and Y179F showed no significant contribution for Tyr-415 to substrate binding and only a small contribution for Tyr-179. Changes in kinetics for T14A, Q47E, and R46A enzymes implicate these residues, to differing extents, in coenzyme binding and discrimination between NADP(+) and NAD(+). By the same measure, Lys-343 is also involved in defining coenzyme specificity. Decrease in k(cat) and k(cat)/K(m) for the D374Q mutant enzyme defines the way Asp-374, unique to L. mesenteroides G6PD, modulates stabilization of the enzyme during catalysis by its interaction with Lys-182. The greatly reduced k(cat) values of enzymes P149V and P149G indicate the importance of the cis conformation of Pro-149 in accessing the correct transition state.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Delineation of the Roles of Amino Acids Involved in the Catalytic Functions of Leuconostoc mesenteroides Glucose 6-Phosphate Dehydrogenase

et al. 2000

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“…In both enzymes, NADPH is a competitive inhibitor against NADP + , whereas NADH is a mixed inhibitor against NAD + [48]. Finally, the use of NAD + over NADP + in glucose-6-phosphate dehydrogenase is promoted by increasing the concentration of glucose-6-phosphate [49]. The preference of P. aeruginosa BADH for the coenzyme used also seems to be affected by the concentration of betaine aldehyde, although to a smaller extent, as suggested by the value of k cat \K betaine aldehyde m , which is 25 % higher in the NAD + -dependent than in the NADP + -dependent reaction (Table 1).…”

Section: Physiological Implications Of the Kinetic Mechanismmentioning

confidence: 99%

Steady-state kinetic mechanism of the NADP+- and NAD+-dependent reactions catalysed by betaine aldehyde dehydrogenase from Pseudomonas aeruginosa

2000

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Betaine aldehyde dehydrogenase (BADH) catalyses the irreversible oxidation of betaine aldehyde to glycine betaine with the concomitant reduction of NAD(P)(+) to NADP(H). In Pseudomonas aeruginosa this reaction is a compulsory step in the assimilation of carbon and nitrogen when bacteria are growing in choline or choline precursors. The kinetic mechanisms of the NAD(+)- and NADP(+)-dependent reactions were examined by steady-state kinetic methods and by dinucleotide binding experiments. The double-reciprocal patterns obtained for initial velocity with NAD(P)(+) and for product and dead-end inhibition establish that both mechanisms are steady-state random. However, quantitative analysis of the inhibitions, and comparison with binding data, suggest a preferred route of addition of substrates and release of products in which NAD(P)(+) binds first and NAD(P)H leaves last, particularly in the NADP(+)-dependent reaction. Abortive binding of the dinucleotides, or their analogue ADP, in the betaine aldehyde site was inferred from total substrate inhibition by the dinucleotides, and parabolic inhibition by NADH and ADP. A weak partial uncompetitive substrate inhibition by the aldehyde was observed only in the NADP(+)-dependent reaction. The kinetics of P. aeruginosa BADH is very similar to that of glucose-6-phosphate dehydrogenase, suggesting that both enzymes fulfil a similar amphibolic metabolic role when the bacteria grow in choline and when they grow in glucose.

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“…G6PDHs from barley and Arabidopsis are specific for NADP + but not NAD + [15,16]. SCG is a NADP + -preferring enzyme, while LMG catalyzes both NAD + -and NADP + -linked reactions [44,45]. Here, His-G6PDH was found to be a NADP + -specific enzyme (Fig.…”

Section: Discussionmentioning

confidence: 76%

“…Kinetic properties of G6PDH from potato [17], LMG [44], and SCG [45] were investigated at 25℃. It was reported that even 26℃ inhibited growth and development of tomato [46].…”

Section: Discussionmentioning

confidence: 99%

Application of Solanum lycopersicum Glucose-6-phosphate Dehydrogenase to NADPH-generating System for Cytochrome P450 Reactions

Park¹,

Jeong²,

Hoon³

et al. 2019

Microbiology and Biotechnology Letters

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Cytochrome P450 (P450 or CYP) is involved in the metabolism of endogenous and exogenous compounds in most organisms. P450s have great potential as biocatalysts in the pharmaceutical and fine chemical industries because they catalyze diverse oxidative reactions using a wide range of substrates. The high-cost nicotinamide cofactor, NADPH, is essential for P450 reactions. Glucose-6-phosphate dehydrogenase (G6PDH) has been commonly used in NADPH-generating systems (NGSs) to provide NADPH for P450 reactions. Currently, only two G6PDHs from Leuconostoc mesenteroides and Saccharomyces cerevisiae can be obtained commercially. To supply high-cost G6PDH cost-effectively, we cloned the cytosolic G6PDH gene of Solanum lycopersicum (tomato) with 6xHis tag, expressed it in Escherichia coli, and purified the recombinant G6PDH (His-G6PDH) using affinity chromatography. In addition, enzymatic properties of His-G6PDH were investigated, and the His-G6PDH-coupled NGS was optimized for P450 reactions. His-G6PDH supported CYP102A1-catalyzed hydroxylation of omeprazole and testosterone by NADPH generation. This result suggests that tomato His-G6PDH could be a cost-effective enzyme source for NGSs for P450-catalyzed reactions as well as other NADPH-requiring reactions.

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Simultaneous analysis of NAD- and NADP-linked activities of dual nucleotide-specific dehydrogenases. Application to Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase.

Cited by 26 publications

References 16 publications

Delineation of the Roles of Amino Acids Involved in the Catalytic Functions of Leuconostoc mesenteroides Glucose 6-Phosphate Dehydrogenase

Delineation of the Roles of Amino Acids Involved in the Catalytic Functions of Leuconostoc mesenteroides Glucose 6-Phosphate Dehydrogenase

Steady-state kinetic mechanism of the NADP+- and NAD+-dependent reactions catalysed by betaine aldehyde dehydrogenase from Pseudomonas aeruginosa

Application of Solanum lycopersicum Glucose-6-phosphate Dehydrogenase to NADPH-generating System for Cytochrome P450 Reactions

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