American Journal of Advanced Drug Delivery

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Original Article Pyrroquinoline as an In-vivo Biofactor in Polyol Pathway Dr. Dhrubo Jyoti Sen* and Dr. R. Badmanaban

Department of Pharmaceutical Chemistry and Phytochemistry, Shri Sarvajanik Pharmacy College, Gujarat Technological University, Arvind Baug, Mehsana-384001, Gujarat, India

Date of Receipt- 09/12/2014 ABSTRACT Date of Revision- 10/12/2014 Date of Acceptance- 15/12/2014 Pyrroquinoline quinone-dependent (PQQ-ADH) of acetic acid bacteria is a membrane-bound involved in the acetic acid fermentation by oxidizing ethanol to acetaldehyde coupling with reduction of membranous ubiquinone (Q), which is, in turn, re- oxidized by ubiquinol oxidase, reducing oxygen to water. PQQ-ADHs

seem to have co-evolved with the organisms fitting to their own habitats.

The enzyme consists of three subunits and has a pyrroloquinoline quinone, 4 c moieties and a tightly bound Q as the electron transfer mediators. Biochemical, genetic and electrochemical studies have revealed the unique properties of PQQ-ADH since it was purified in 1978. The enzyme is unique to have ubiquinol oxidation activity in addition to Q reduction. This focuses on the molecular properties of PQQ-ADH, such as the roles of the subunits and the cofactors, particularly in intramolecular electron transport of the enzyme from ethanol to Q. Biotechnological applications of PQQ-ADH as to

enantiospecific oxidations for production of the valuable chemicals and bioelectrocatalysis for sensors and fuel cells using indirect and direct Address for electron transfer technologies and discuss unsolved issues and future Correspondence prospects related to this elaborate enzyme. PQQ with ABCDE gene cluster from Gluconobacter oxydans, which is involved in Department of pyrroloquinoline quinone (PQQ) biosynthesis, in Escherichia coli, Pharmaceutical resulting in PQQ accumulation in the medium. Since the gene cluster Chemistry and does not include the tldD gene needed for PQQ production, this result Phytochemistry, Shri suggests that the E. coli tldD gene, which shows high homology to the Sarvajanik Pharmacy G. oxydans tldD gene, carries out that function. The synthesis of PQQ College, Gujarat activated d-glucose dehydrogenase in E. coli and the growth of the Technological recombinant DNA has been improved. In an attempt to increase the University, Arvind production of PQQ, which acts as a or growth factor, E. coli has Baug, Mehsana- been transferred with various recombinant plasmids, resulting in the 384001, Gujarat, India. overproduction of the PQQ synthesis and, consequently, PQQ E-mail: dhrubosen69 accumulation--up to 6 mM--in the medium. @yahoo.com Keywords: PQQ, ADH, Q10, Polyol, NADPH, NAD+, E. coli.

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INTRODUCTION Pyrroloquinoline quinone (PQQ) was NADH from NAD+. Hexokinase can return discovered by J.G. Hauge as the third redox the molecule to the glycolysis pathway by after and flavin in phosphorylating fructose to form fructose-6- bacteria (although he hypothesized that it phosphate. However, in uncontrolled was naphthoquinone).1 Anthony and Zatman diabetics that have high blood glucose - more also found the unknown redox cofactor in than the glycolysis pathway can handle - the alcohol dehydrogenase and named it reaction's mass balance ultimately favors the methoxatin. In 1979, Salisbury and production of sorbitol.7 (See figure 2.) colleagues as well as Duine and colleagues extracted this from Mode of action of methylotrophs Activation of the polyol pathway and identified its molecular structure. results in a decrease of reduced NADPH and Adachi and colleagues identified that PQQ oxidized NAD+; these are necessary cofactors was also found in Acetobacter.2 (See figure in redox reactions throughout the body and 1.) under normal conditions they are not The previous publication of Q10 was interchangeable.8 The decreased concen- on cholesterol lowering device and present tration of these NADPH leads to decreased article focuses on glucose lowering activity synthesis of reduced , nitric oxide, in-vivo. [Piyush A. Gediya, Nadim M. myo- inositol, and taurine. Myo-inositol is Chhipa, Viraj P. Jatakiya, Sachin M. Patel particularly required for the normal function and Prof. Dr. Dhrubo Jyoti Sen; of nerves. Sorbitol may also glycate nitrogens as free radical scavengers in on proteins, such as collagen and the products hypercholesterolemia: Internationale Phar- of these glycations are referred-to as AGEs - maceutica Sciencia: 2(4), 18-25, 2012.]3 advanced glycation end products.9 (See figure These enzymes containing PQQ are 3) called quinoproteins. Glucose dehydrogenase, AGEs are thought to cause disease in one of the quinoproteins, is used as a glucose the human body, one effect of which is sensor. Subsequently, PQQ was found to mediated by RAGE (Receptor for Advanced stimulate growth in bacteria.4 In addition, Glycation End products) and the ensuing antioxidant and neuro-protective effects were inflammatory responses induced.10 They are also found. Also called the sorbitol-aldose seen in the hemoglobin A1C tests performed reductase pathway, the polyol pathway on known diabetics to assess their levels of appears to be implicated in diabetic glucose control. Aldose reductase inhibitors complications, especially in microvascular are a class of drugs being studied as a way to damage to the retina, kidney and nerves. prevent eye and nerve damage in people with Sorbitol cannot cross cell membranes and diabetes. Their target, aldose reductase, is an when it accumulates, it produces osmotic enzyme that is normally present in many stresses on cells by drawing water into the other parts of the body and catalyzes one of insulin-independent tissues.5 (See table 1.) the steps in the sorbitol (polyol) pathway that Cells use glucose for energy; is responsible for fructose formation from however, unused glucose enters the polyol glucose.11 Aldose reductase activity increases pathway when aldose reductase reduces it to as the glucose concentration rises in diabetes sorbitol.6 This reaction oxidizes NADPH to in those tissues that are not insulin sensitive, NADP+. Sorbitol dehydrogenase can then which include the lenses, peripheral nerves oxidize sorbitol to fructose, which produces and glomerulus. Sorbitol does not diffuse

AJADD[2][6][2014] 816-823 Sen et al______ISSN 2321-547X through cell membranes easily and therefore CONCLUSION accumulates, causing osmotic damage which leads to retinopathy and neuropathy. While Pyrroloquinoline quinone (PQQ) is a most cells require the action of insulin for novel biofactor for which a proposition can be glucose to gain entry into the cell, the cells of made for physiological importance. PQQ was the retina, kidney and nervous tissues are first recognized as an enzyme cofactor in insulin-independent, so glucose moves freely bacteria. It has recently been tentatively across the cell membrane, regardless of the identified as a component of interstellar dust. action of insulin.12 Thus, PQQ may have been present throughout The cells will use glucose for energy early biological conception and evolution. as normal and any glucose not used for PQQ is also a potent plant growth factor. energy will enter the polyol pathway. When Consequently, for animals and humans, there blood glucose is normal (about 100 mg/dl or has been constant exposure to PQQ. In 5.5 mmol/l), this interchange causes no animals, PQQ is reported to participate in a problems, as aldose reductase has a low range of biological functions with apparent affinity for glucose at normal concen- survival benefits (e.g., improved neonatal trations.13 In a hyperglycemic state, the growth and reproductive performance). There affinity of aldose reductase for glucose rises, are also benefits from PQQ supplementation causing much sorbitol to accumulate, and related to cognitive, immune, and antioxidant using much more NADPH, leaving less functions, as well as protection from cardiac NADPH for other processes of cellular and neurological ischemic events. Although metabolism. This change of affinity is what is PQQ is not currently viewed as a vitamin, its meant by activation of the pathway. The involvement in cell signaling pathways, amount of sorbitol that accumulates, however, particularly those important to may not be sufficient to cause osmotic influx mitochondriogenesis in experimental animal of water.14 NADPH acts to promote nitric models, may eventually provide a rationale oxide and glutathione production, and its for defining PQQ as vital to life. For humans, deficiency will cause glutathione deficiency such evidence suggests there may be similar as well. A glutathione deficiency, congenital parallels or benefits from improving PQQ or acquired, can lead to hemolysis caused by status. (See figure 4 & 5.) oxidative stress.15 Nitric oxide is one of the The observation that increased important vasodilators in blood vessels.16 mitochondriogenesis and antioxidant Therefore NADPH prevents reactive oxygen functions may be healthful features of PQQ species from accumulating and damaging supplementation opens the doors for both cells. Excessive activation of the polyol therapeutic applications and possible use as pathway increases intracellular and an ergogenic aid. Having normal extracellular sorbitol concentrations, mitochondrial function is essential to a broad increased concentrations of reactive oxygen range of health and disease relationships; species and decreased concentrations of nitric thus, the need for continuing research that oxide and glutathione. Each of these examines the efficacy and use of PQQ is imbalances can damage cells; in diabetes compelling. PQQ derivatives are widely there are several acting together. It has not distributed in tissues and biological fluids at been conclusively determined that activating concentrations that may be sustained by the polyol pathway damages micro- typical dietary exposures. Given the range of vasculature.17 functions and apparent survival benefits (e.g., improved reproductive performance), it is reasonable to suggest that PQQ may play a

AJADD[2][6][2014] 816-823 Sen et al______ISSN 2321-547X fundamental role in metabolism. Diabetic affinity transport system of unknown identity, cataract formation follows an increase in followed most likely by phosphorylation via sugars in the lens. The excess sugar within the glucokinase. lens is reduced by aldose reductase to its alcohol, but the lens capsule is relatively REFERENCES impermeable to sugar alcohols. Because of the excess sugar alcohol (polyol), the lens 1. Hauge JG. Glucose dehydrogenase of imbibes water, causing osmotic imbalance. bacterium anitratum: an enzyme with a Eventually, increased sodium and decreased novel prosthetic group. J Biol Chem. potassium levels and decreased glutathione 1964; 239:3630–3639. levels lead to cataract formation. Topical 2. Anthony C, Zatman LJ. The microbial administration of aldose reductase inhibitors oxidation of methanol. The prosthetic prevents the cataract in rats. Many bacteria group of the alcohol dehydrogenase of can synthesize the cofactor pyrroloquinoline Pseudomonas sp. M27: a new oxido- quinone (PQQ), a cofactor of several reductase prosthetic group. Biochem dehydrogenases, including glucose J. 1967; 104(3):960–969. dehydrogenase (GCD). Among the enteric 3. Gediya PA, Chhipa NM, Viatakiya VP, bacteria, Klebsiella pneumoniae has been Patel SM and Sen DJ. Antioxidant shown to contain the genes required for PQQ coenzyme Q10 as free radical biosynthesis. Escherichia coli and Salmonella scavengers in hypercholesterolemia: typhimurium were thought to be unable to Internationale Pharmaceutica Sciencia. synthesize PQQ but it has been reported that 2012; 2(4):18-25. strain EF260, a derivative of E. coli FB8, can 4. Salisbury SA, Forrest HS, Cruse WB, synthesize PQQ after mutation and can Kennard O. A novel coenzyme from oxidize glucose to gluconate via the bacterial primary alcohol dehydro- GCD/PQQ pathway (F. Biville, E. Turlin & genases. Nature. 1979; 280(5725):843– F. Gasser, 1991, J Gen Microbiol 137, 1775- 844. 1782). We have re-investigated this claim and 5. Westerling J, Frank J, Duine JA. The conclude that it is most likely erroneous. (i) prosthetic group of methanol Strain EF260, isolated originally by Biville dehydrogenase from Hyphomicrobium and coworkers, was unable to synthesize a X: electron spin resonance evidence for holo-enzyme GCD unless PQQ was supplied a quinone structure. Biochem Biophys to the growth medium. No GCD activity Res Commun. 1979; 87(3):719–724. could be detected in membrane fractions. (ii) 6. Ameyama M, Matsushita K, Ohno Y, The amount of PQQ detected in the growth Shinagawa E, Adachi O. Existence of a medium of EF260 was very low and not very novel prosthetic group, PQQ, in different from that found in a medium with its membrane-bound, electron transport parent strain or in a medium containing no chain-linked, primary dehydrogenases of cells. (iii) EF260 cells were unable to produce oxidative bacteria. FEBS Lett. 1981; gluconate from glucose via the PQQ/GCD 130(2):179–183. pathway. (iv) Introduction of a gcd:: Cm 7. Ameyama M, Matsushita K, Shinagawa deletion in EF260, eliminating GCD, did not E, Hayashi M, Adachi O. affect glucose metabolism. This suggested a Pyrroloquinoline quinone: excretion by pathway for glucose metabolism other than methylotrophs and growth stimulation the PQQ/GCD pathway. (v) Glucose uptake for microorganisms. Bio Factors. 1988; and metabolism in EF260 involved a low- 1(1):51–53.

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8. Rucker R, Chowanadisai W, Nakano M. modulatory site. Journal of Neuro- Potential physiological importance of science. 1992; 12(6):2362–2369. pyrroloquinoline quinone. Altern Med 13. Aizenman E, Jensen FE, Gallop PM, Rev. 2009; 14(3):179–183. Rosenberg PA, Tang LH. Further 9. Chowanadisai W, Bauerly KA, evidence that pyrroloquinoline quinone Tchaparian E, Wong A, Cortopassi GA, interacts with the N-methyl-D-aspartate Rucker RB. Pyrroloquinoline quinone receptor redox site in rat cortical neurons stimulates mitochondrial biogenesis in vitro. Neuroscience letters. 1994; 168 through cAMP response element- (1-2):189–192. binding protein phosphorylation and 14. Scanlon JM, Aizenman E, Reynolds IJ. increased PGC-1alpha expression. Effects of pyrroloquinoline quinone on Journal of Biological Chemistry. 2010; glutamate-induced production of 285(1):142–152. reactive oxygen species in neurons. 10. Lanza IR, Sreekumaran Nair European Journal of Pharmacology. K. Regulation of skeletal muscle 1997; 326(1):67–74. mitochondrial function: genes to 15. Hossain MA. Molecular mediators of proteins. Acta Physiologica. 2010; hypoxic-ischemic injury and 199(4):529–547. implications for epilepsy in the 11. Zhang Y, Feustel P, Kimelberg H. developing brain. Epilepsy & Behavior. Neuroprotection by pyrroloquinoline 2005; 7(2):204–213. quinone (PQQ) in reversible middle 16. Felton LM, Anthony C. Biochemistry: cerebral artery occlusion in the adult role of PQQ as a mammalian enzyme rat. Brain Research. 2006; 1094(1):200– cofactor? Nature. 2005; 433(7025):E10; 206. discussion E11–12. 12. Aizenman E, Hartnett KA, Zhong C, 17. Rucker R, Chowanadisai W and Nakano Gallop PM, Rosenberg PA. Interaction M. Potential Physiological Importance of the putative essential nutrient of Pyrroloquinoline Quinone. Alternative pyrroloquinoline quinone with the N- Medicine Review. 2009; 14(3):268-277. methyl-D-aspartate receptor redox

Table 1. Physical properties of PQQ2

Properties Value Properties Value LogP -0.65 Parachor 573.1 ± 6.0 cm3

Molecular Formula C14H6N2O8 Index of Refraction 1.801 ± 0.02 Formula Weight 330.20604 Surface Tension 134.8 ± 3.0 dyne/cm Composition C(50.92%) H(1.83%) N(8.48%) O(38.76%) Density 1.963 ± 0.06 g/cm3 Molar Refractivity 71.98 ± 0.3 cm3 Polarizability 28.53 ± 0.5 10-24cm3 Molar Volume 168.2 ± 3.0 cm3 Monoisotopic Mass 330.012415 Da

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O

OH O OH NH

HO N O

O O 4,5-dioxo-4,5-dihydro-1H-pyrrolo[2,3-f]-2,7,9-tricarboxylic acid

Figure 1. Pyrroquinoline quinone1,2

Figure 2. Poly ol pathway3,4

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4,5-dihydroxy-1H -pyrrolo[2,3-f]quinoline-2,7,9-tricarboxylic acid O O O H O H O O H O O H N H N H Oxidase/Reductase E nzyme

H O H O N O N O H

O O O O H 4,5 -diox o-4,5-dihydro-1H -pyrrolo[2,3-f]quinoline-2,7,9-tricarbox ylic acid

Figure 3. PQQ as cofactor5,6

16 Figure 4. PQQ formulation

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- - O + O + - O + O NH N BF N N 2 2 4 Balz-Schiemann Reaction Ac2O, HCOOH H2, Pd (C) Acetylation Reduction NH NH NH NH2 O O O O H C O H H3C O H H3C O H 3 H3C O

Acetylation H3C O

CH2

H3C O

CH CH CH2 3 3 O O O O O Reduction O CH3 O H N N O H NH N O N 2 HN H H

CH2 CH2 Fischer Indole Synthesis O O NH

H3C CH3 O O O H3C O H O O Conrad-Limpach Synthesis O H2C O O O H2C O NH CH3 O O (NH4)2Ce(NO3)6 NH CH3 Oxidation O N O O N O O H3C H3C O O H3C

Hydrolysis O H2C O O OH NH

O O N

O OH

Figure 5. PQQ Synthetic route7-10

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