Quinate Oxidation Membrane Bound Quinate Dehydrogenase QDH

Quinate ^ 3-dehydroquinate

Unlike enzymes relating to the shikimate pathway found in the cytoplasm of microorganisms, the first report of a quinate-oxidizing enzyme in acetic acid bacteria was done by Whiting and Coggins [76]. They described quinate oxidation to 3-dehydroquinate (DQA) by an NAD (P)-independent quinate dehydrogenase (QDH) (EC and shikimate (SKA) oxidation to 3-dehydroshikimate (DSA) by an enzyme associated with the particulate enzyme in the cytoplasmic membrane. van Kleef and Duine described the occurrence of QDH in the periplasm of Acinetobacter calcoaceticus LMD 79.41 and suggested that QDH is a quinprotein in which PQQ is involved [77] (Fig. 1.8).

QDH purification was done with Gluconobacter oxydans IFO 3244 and Acinetobacter calcoaceticus AC3 after solubilization with detergent [78, 79]. Due to the hydrophobicity of QDH as a typical membrane-bound enzyme, QDH is one of the most difficult enzymes to purify to high homogeneity. The molecular mass of QDH was estimated to be 88 kDa. It oxidizes quinate and SKA with optimal activity at pH 6-7. Since A. calcoaceticus AC3 cannot produce PQQ, QDH was

Shikimate Pathway
Fig. 1.8 Metabolic map of shikimate pathway.

purified as an apoenzyme consisting of dimeric structure, which was converted to the monomeric holoenzyme on addition of PQQ.

QDH is very beneficial enzyme for the production of SKA via DQA and DSA from quinate supplied outside the SKA pathway without any metabolic control repressing SKA production. In 2003, we proposed SKA production by a single cellular system of acetic acid bacteria [80-83]. QDH and 3-dehydroquinate dehydratase (DQD) are located predominantly on the outer surface of the cytoplasmic membranes of some species of Gluconobacter strains and quinate is oxidized to DSA via DQA in a sequential manner. In the cytoplasm, SKDH catalyzes a reversible reaction of SKA oxidation to DSA and DSA reduction to SKA. However, many trial and error experiments to combine together the two separately located enzymatic systems taking place outside and inside the cells only resulted in insufficient production of SKA.

As a more positive response to the global need for oseltamivir, we have developed a better strategy for high SKA production, allowing the two separately located enzymatic systems to work sequentially. Dried cells or the membrane fraction involving QDH and DQD may be used for DSA production in the first reaction (system 1 in Fig. 1.9a). The second reaction is a coupling reaction composed of two cytosolic enzymes, SKDH and GDH, as an NADPH-regenerating enzyme (system 2 in Fig. 1.9a). The coupling reaction by the two cytosolic enzymes in the presence of excess D-glucose works well as expected until the DSA added initially is converted completely to SKA. The overall reaction carried out for SKA production by the two different enzymatic systems is depicted in Fig. 1.9b.

SKA is a key intermediate for aromatic amino acids as well as for large numbers of antibiotics, alkaloids, and herbicides. Recently, another important role for SKA has emerged as a precursor for the synthesis of oseltamivir (Tamiflu), an antiviral drug designed to protect people from pandemic flu infection. In spite of warnings from the World Health Organization about the approach of a global flu pandemic, including avian influenza, there are insufficient stocks of oseltamivir around the world. One reason for this is the technical difficulties in preparing SKA, because two different metabolic pathways, glycolysis and pentose phosphate pathway, need to be combined before forming SKA. Furthermore the metabolic location of SKA is a long way from that for glucose and it is very difficult to lead the metabolic flow to SKA production through classic fermentation technology as well as through modern molecular biotechnology. Although resources are limited, SKA is also produced by extraction from plants such as Illicium anisatum or I. verum. The total synthesis of SKA through organic chemistry has not been practically available. Nevertheless, we need to address the challenge of developing a novel method for more effective and convenient SKA production.

SKA is remote from D-glucose in the metabolic pathway, the more shortcut access to the SKA pathway from quinate looks advantageous to produce the important metabolic intermediates generated in the SKA pathway. DSA is formed with a yield of about 90% from quinate via DQA by two suc-cessive enzyme reactions, QDH and 3-dehydroquinate dehydratase (DQD) (EC located in the

(a) System 1 : Oxidative fermentation for 3-dehydroshikimate formation from quinate

50 100 150 200 250 Incubation time (min)

Fig. 1.9 (a) Overall reaction for SKA production and (b) enzymatic conversion of DSA to SKA with SKDH coupled with GDH.

cytoplasmic membranes of acetic acid bacteria. DSA is then reduced to SKA with NADP-dependent SKA dehydrogenase (SKDH) (EC from the same organism. When SKDH is coupled with NADP-dependent glucose dehydrogenase (GDH) (EC in the presence of excess D-glucose as an NADPH regenerating system, SKDH works to produce SKA until DSA added initially in the reaction mixture is completely converted to SKA as shown in Fig. 1.9b [83, 84].

For the system 1, the dried cells or the membrane fraction were incubated with quinate at pH 5.0 overnight with shaking. The DSA formed was measured with the deproteinized supernatants. Since DQD have a pH optimum at 8.0 and low activity of DQD is found at pH 5.0, the reaction can be regulated to make DQA as the major product. The apparent conversion rate from quinate to DQA is estimated at over 90%. On the other hand, when the above reaction was carried out at pH 8.0, DSA was accumulated as the major product, because DQA formed can be converted immediately to DSA under the optimum condition of DQD. The apparent yield of DSA from quinate is usually over 90% [85]. Thus, a convincing strategy for preparing commercially unavailable metabolic intermediates, DQA and DSA, by means of bioconversion has been established. QDA is one of the compounds involved in the oxidative fermentation, the initially added quinate is converted to the corresponding oxidation product with high yield, as seen with other PQQ- and FAD-dependent dehydrogenases. A simple chromatographic method for separating the four compounds - quinate, DQA, DSA, and SKA - in the SKA pathway has recently become available [85].

Was this article helpful?

0 0
Heal Yourself With Qi Gong

Heal Yourself With Qi Gong

Qigong also spelled Ch'i Kung is a potent system of healing and energy medicine from China. It's the art and science of utilizing breathing methods, gentle movement, and meditation to clean, fortify, and circulate the life energy qi.

Get My Free Ebook

Post a comment