Structure And Mutagenic Conversion of E(1) Dehydrase: at the Crossroads of Dehydration, Amino Transfer, And Epimerization
Pyridoxal 5'-phosphate (PLP) and pyridoxamine 5'-phosphate (PMP) are highly versatile coenzymes whose importance is well recognized. The capability of PLP/PMP-dependent enzymes to catalyze a diverse array of chemical reactions is attributed to fine-tuning of the cofactor-substrate interactions in the active site. CDP-6-deoxy-l-threo-d-glycero-4-hexulose 3-dehydrase (E1), along with its reductase (E{sub 3}), catalyzes the C-3 deoxygenation of CDP-4-keto-6-deoxy-d-glucose to form the dehydrated product, CDP-4-keto-3,6-dideoxy-d-glucose, in the ascarylose biosynthetic pathway. This product is the progenitor to most 3,6-dideoxyhexoses, which are the major antigenic determinants of many Gram-negative pathogens. The dimeric [2Fe-2S] protein, E{sub 1}, cloned from Yersinia pseudotuberculosis, is the only known enzyme whose catalysis involves the direct participation of PMP in one-electron redox chemistry. E{sub 1} also contains an unusual [2Fe-2S] cluster with a previously unknown binding motif (C-X{sub 57}-C-X{sub 1}-C-X{sub 7}-C). Herein we report the first X-ray crystal structure of E{sub 1}, which exhibits an aspartate aminotransferase (AAT) fold. A comparison of the E{sub 1} active site architecture with homologous structures uncovers residues critical for the dehydration versus transamination activity. Site-directed mutagenesis of four E{sub 1} residues, D194H, Y217H, H220K, and F345H, converted E{sub 1} from a PMP-dependent dehydrase to a PLP/glutamate-dependent aminotransferase. The E{sub 1} quadruple mutant, having been conferred this altered enzyme activity, can transaminate the natural substrate to CDP-4,6-dideoxy-4-amino-d-galactose without E{sub 3}. Taken together, these results provide the molecular basis of the functional switch of E{sub 1} toward dehydration, epimerization, and transamination. The insights gained from these studies can be used for the development of inhibitors of disease-relevant PLP/PMP-dependent enzymes.
- Research Organization:
- SLAC National Accelerator Lab., Menlo Park, CA (United States)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC02-76SF00515
- OSTI ID:
- 953569
- Report Number(s):
- SLAC-REPRINT-2009-314; BICHAW; TRN: US201002%%1397
- Journal Information:
- Biochem. 47:6329,2008, Vol. 47, Issue 24; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
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