A Retro-Evolution Study of CDP-6-deoxy-d-glycero-l-threo-4-hexulose-3-dehydrase (E₁) from Yersinia pseudotuberculosis:  Implications for C-3 Deoxygenation in the Biosynthesis of 3,6-Dideoxyhexoses

Abstract

CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E 1 ), which catalyzes C-3 deoxygenation of CDP-4-keto-6-deoxyglucose in the biosynthesis of 3,6-dideoxyhexoses, shares a modest sequence identity with other B 6 -dependent enzymes, albeit with two important distinctions. It is a rare example of a B 6 -dependent enzyme that harbors a [2Fe-2S] cluster, and a highly conserved lysine that serves as an anchor for PLP in most B 6 -dependent enzymes is replaced by histidine at position 220 in E 1 . Since alteration of His220 to a lysine residue may produce a putative progenitor of E 1 , the H220K mutant was constructed and tested for the ability to process the predicted substrate, CDP-4-amino-4,6-dideoxyglucose, using PLP as the coenzyme. Our data showed that H220K-E 1 has no dehydrase activity, but can act as a PLP-dependent transaminase. However, the reaction is not catalytic since PLP cannot be regenerated during turnover. Reported herein are the results of this investigation and the implications for the role of His220 in the catalytic function and mechanism of E 1 .The C-3 deoxygenation step in the biosynthesis of 3,6-dideoxyhexoses, in which CDP-4-keto-6-deoxy-D-glucose (1) is converted to CDP-4-keto-3,6-dideoxy-D-glucose (2), is catalyzed by CDP-6-deoxy-D-glycero-L-threo-4-hexulose-3-dehydrase (E 1 ) and a reductase (E 3 ). 1-4 E 1 is a dimeric protein containing one pyridoxamine 5′-phosphate (PMP) and a [2Fe-2S] cluster per subunit. 5-7 E 3 belongs to the flavodoxin-NADP + reductase family and contains FAD and a plant-type ferredoxin [2Fe-2S] center in the active site. 8,9 The reaction catalyzed by E 1 and E 3 is initiated by the formation of a Schiff base between PMP and the 4-keto group of 1 (Scheme 1). 5,6 Subsequent abstraction of the pro-S 4′-H of the external Schiff base (3) results in the loss of the 3-OH group and leads to the conjugated intermediate (4). 1-7 A sequential two-electron reduction relayed from NADH via E 3 -bound FAD and the [2Fe-2S] centers of E 1 and E 3 drives the reaction to completion and regenerates the PMP coenzyme. 10,11 A radical intermediate represented by 5 has been established by EPR analysis during turnover. 12,13 The participation of PMP in deoxygenation is unusual, as is the direct

Stereospecific synthesis of sugar-1-phosphates and their conversion to sugar nucleotides

“…the coupling step helped to minimize these byproducts, but could not completely suppress their formation. In the case of the D D-glucose (20,21) and L L-arabinosecontaining (26,27) sugar nucleotides, tributylammonium acetate was used as an ion-pair reagent (10 mM aqueous buffer at pH 4) in C18 reversed-phase ion-pair chromatography as previously described for the purification of nucleoside 5 0 -b, c-methylenetriphosphate analogues. 35 Interestingly, this ion-pair reagent did not permit the separation of L L-rhamnose (22,23) and L L-fucose-containing (24,25) sugar nucleotides from dinucleoside diphosphate byproducts.…”

“…24 Although improved yields have been reported using 1H-tetrazole as a catalyst 25 or through extensive co-evaporations with pyridine, 26 many coupling reactions of this type take several days to reach completion and result in only low to moderate yields (<50%) following purification. [27][28][29][30][31] Herein, we report the synthesis and purification of eight sugar nucleotides via coupling of sugar-1-phosphates with nucleoside 5 0 -monophosphates activated using trifluoroacetic anhydride and N-methylimidazole. This activation method was first reported by Bogachev for use in the synthesis of nucleoside 5 0 -triphosphates, 32 and has since been used in the synthesis of UDP-a-D Dgalactofuranose by Marlow and Kiessling, 33 for the preparation of GDP-hexanolamine by Vincent and Gastinel 34 and in the synthesis of electron-deficient nucleoside 5 0 -b,c-methylenetriphosphate analogues by Mohamady and Jakeman.…”

Mechanisms and structures of vitamin B6-dependent enzymes involved in deoxy sugar biosynthesis

“…The most obvious residue in E 1 that is incongruent with common PLP-dependent aminotransferases is the histidine found in place of the strictly conserved lysine present in all PLP-containing enzymes. The conserved histidine found in place of this residue was mutated to lysine (H220K mutant) which immediately endowed E 1 with aminotransferase activity, allowing production of CDP-4-amino-4,6-dideoxy-D-glucose using the same substrate of the wild type E 1 reaction, CDP-4-keto-6-deoxy-D-glucose ( 68 ) [94]. However, this reaction was non-catalytic, as only one round of reaction was possible because the PLP cofactor could not be regenerated.…”

A Structural Study of GDP-4-Keto-6-Deoxy-d-Mannose-3-Dehydratase: Caught in the Act of Geminal Diamine Formation,

“…It catalyzes the removal of the sugar C3′-hydroxyl group from many CDP-linked 3,6-dideoxyhexoses (19). Although E 1 requires E 3 (with its accompanying cofactors) for activity, it is strikingly similar to ColD in that the typical active site lysine is replaced with a histidine.…”

“…Interestingly, it was shown that changing this histidine in E 1 to a lysine residue via site-directed mutagenesis resulted in an enzyme with no dehydratase activity. The enzyme was, however, capable of performing a one turnover event of the aminotransferase reaction (19).…”