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