Evolution of Enzymatic Activities in the Enolase Superfamily:  l-Fuconate Dehydratase from Xanthomonas campestris^,

Abstract

Many members of the mechanistically diverse enolase superfamily have unknown functions. In this report we use both genome (operon) context and screening of a library of acid sugars to assign the L-fuconate dehydratase (FucD) function to a member of the mandelate racemase (MR) subgroup of the superfamily encoded by the Xanthomonas campestris pv. campestris str. ATCC 33913 genome (GI:21233491). Orthologues of FucD are found in both bacteria and eukaryotes, the latter including the rTS beta protein in Homo sapiens that has been implicated in regulating thymidylate synthase activity. As suggested by sequence alignments and confirmed by high-resolution structures in the presence of active site ligands, FucD and MR share the same active site motif of functional groups: three carboxylate ligands for the essential Mg2+ located at the ends of the third, fourth, and fifth beta-strands in the (beta/alpha)7beta-barrel domain (Asp 248, Glu 274, and Glu 301, respectively), a Lys-x-Lys motif at the end of the second beta-strand (Lys 218 and Lys 220), a His-Asp dyad at the end of the seventh and beta-strands (His 351 and Asp 324, respectively), and a Glu at the end of the eighth beta-strand (Glu 382). The mechanism of the FucD reaction involves initial abstraction of the 2-proton by Lys 220, acid catalysis of the vinylogous beta-elimination of the 3-OH group by His 351, and stereospecific ketonization of the resulting enol, likely by the conjugate acid of Lys 220, to yield the 2-keto-3-deoxy-L-fuconate product. Screening of the library of acid sugars revealed substrate and functional promiscuity: In addition to L-fuconate, FucD also catalyzes the dehydration of L-galactonate, D-arabinonate, D-altronate, L-talonate, and D-ribonate. The dehydrations of L-fuconate, L-galactonate, and D-arabinonate are initiated by abstraction of the 2-protons by Lys 220. The dehydrations of L-talonate and D-ribonate are initiated by abstraction of the 2-protons by His 351; however, protonation of the enediolate intermediates by the conjugate acid of Lys 220 yields L-galactonate and D-arabinonate in competition with dehydration. The functional promiscuity discovered for FucD highlights possible structural mechanisms for evolution of function in the enolase superfamily.

Loss of quaternary structure is associated with rapid sequence divergence in the OSBS family

“…S1). Like most other members of the enolase superfamily, the five enzymes from the NSAR/OSBS subfamily are multimers (21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40). The three previously characterized NSAR/OSBS subfamily enzymes are (53).…”

Bifidobacterium longum subsp. infantis ATCC 15697 α-Fucosidases Are Active on Fucosylated Human Milk Oligosaccharides

“…An alternative explanation is that fucose is metabolized via a cryptic oxidative pathway. There is nascent evidence for the existence of an oxidative pathway, based on identification of previously characterized oxidative pathway genes (37). There are two gene clusters within the B. longum subsp.…”

“…One is localized to the HMO cluster (Blon_2337-Blon_2340) and is immediately adjacent to the fucosidases encoded by Blon_2335 and Blon_2336. Of the six enzymes involved in the oxidative pathway, there is evidence for the existence of four of them in the ATCC 15697 genome (37). It is possible that some of these enzymes are multifunctional or have yet to be identified.…”

Crystal Structures of Streptococcus suis Mannonate Dehydratase (ManD) and Its Complex with Substrate: Genetic and Biochemical Evidence for a Catalytic Mechanism

“…His311 is positioned within 3.2 Å of C-2-H position, and Tyr325 is within hydrogen-bonding distance (2.4 Å) to the hydroxyl group at C-3 of the substrate. As observed in the structures of other acid sugar dehydratases (18,24), the divalent Mn 2ϩ ion in the complex structure participates in a sixfold coordination with the side chain oxygen atom of Asp310, the thiol group of Cys237, the carboxyl oxygen atom and C-2-OH group of the substrate, and the nitrogen atoms of His199 and His266 (Fig. 3A).…”

“…The results of kinetic analyses (Table 2) with mutant forms of S. suis ManD provided strong evidence to support our hypothesis. Indeed, basic residues frequently participate in the catalytic process of many dehydratases (8,10,13,24), and tyrosine has also been found to play a critical role in dehydration reactions (1,18). Based on functional assays, comparative sequence alignments, structural analysis, and the results of mutagenesis, a mechanism for the ManD-catalyzed reaction in S. suis is proposed (Fig.…”