Characterization and mutagenesis of two novel iron–sulphur cluster pentonate dehydratases

Abstract

We describe here the identification and characterization of two novel enzymes belonging to the IlvD/EDD protein family, the D-xylonate dehydratase from Caulobacter crescentus, Cc XyDHT, (EC 4.2.1.82), and the L-arabonate dehydratase from Rhizobium leguminosarum bv. trifolii, Rl ArDHT (EC 4.2.1.25), that produce the corresponding 2-keto-3-deoxy-sugar acids. There is only a very limited amount of characterization data available on pentonate dehydratases, even though the enzymes from these oxidative pathways have potential applications with plant biomass pentose sugars. The two bacterial enzymes share 41 % amino acid sequence identity and were expressed and purified from Escherichia coli as homotetrameric proteins. Both dehydratases were shown to accept pentonate and hexonate sugar acids as their substrates and require Mg(2+) for their activity. Cc XyDHT displayed the highest activity on D-xylonate and D-gluconate, while Rl ArDHT functioned best on D-fuconate, L-arabonate and D-galactonate. The configuration of the OH groups at C2 and C3 position of the sugar acid were shown to be critical, and the C4 configuration also contributed substantially to the substrate recognition. The two enzymes were also shown to contain an iron-sulphur [Fe-S] cluster. Our phylogenetic analysis and mutagenesis studies demonstrated that the three conserved cysteine residues in the aldonic acid dehydratase group of IlvD/EDD family members, those of C60, C128 and C201 in Cc XyDHT, and of C59, C127 and C200 in Rl ArDHT, are needed for coordination of the [Fe-S] cluster. The iron-sulphur cluster was shown to be crucial for the catalytic activity (kcat) but not for the substrate binding (Km) of the two pentonate dehydratases.

A combined experimental and modelling approach for the Weimberg pathway optimisation

“…With our initial rate experiments, we confirmed the results for the XDH, XLA and XAD previously determined 33,[38][39][40][41] . We observed some variations in kinetic constants, which are most likely due to differences in the assays that were used (e.g., with respect to buffer system, temperature and metal ions) and differences in enzyme-storage conditions 42 .…”

“…Due to a lack of quantitative models, rational pathway design is not widely applied and time-consuming combinatorial approaches are used instead. For the Weimberg pathway of C. crescentus, only the XDH, the XAD and the KDXD had been partially kinetically characterized [38][39][40][41][42] , and although XLAs were characterized, e.g., from Azospirillum brasilense, Haloferax volcanii and partially from C. crescentus 9,10,33,49 , kinetic constants for C. crescentus were not available. As a prerequisite for rational pathway design and detailed kinetic modelling, we herein established purification procedures and kinetic enzyme assays, and in addition developed protocols for effective synthesis and determination of pathway intermediates.…”

“…For the Weimberg pathway in C. crescentus, enzyme kinetic data are available for three of the five enzymes: XDH, XAD and KDXD, and for XAD also the crystal structure was solved [38][39][40][41][42] . However, product inhibition or inhibition by Weimberg intermediates has not been addressed so far and, to our knowledge, no kinetic model for the pathway has been reported.…”

Crystallization and X-ray diffraction analysis of anL-arabinonate dehydratase fromRhizobium leguminosarumbv.trifoliiand aD-xylonate dehydratase fromCaulobacter crescentus

“…The araD gene encoding R. leguminosarum l-arabinonate dehydratase (RlArDHT) and the xylD gene encoding C. crescentus d-xylonate dehydratase (CcXyDHT) were purchased as codon-optimized synthetic genes from GeneArt, Germany and a Strep-Tag II (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) was added at the N-terminus (the deposited GenBank accession numbers for araD and xylD are KT260159 and KT260154, respectively). The genes were cloned into a pBAT4 expression vector, as described in Andberg et al (2016). Other information on macromolecule production is given in Table 1.…”

“…trifolii and d-xylonate dehydratase from Caulobacter crescentus belong to the IlvD/EDD enzyme family. Enzymes in this family contain iron-sulfur [Fe-S] clusters as a prosthetic group (Andberg et al, 2016;Nunn et al, 2010;Stephens et al, 2007) and, using spectroscopic methods, active enzymes from this family have been found to contain either a [4Fe-4S] cluster (Watanabe et al, 2006;Rodriguez et al, 1996;Flint et al, 1993) or a [2Fe-2S] cluster (Flint & Emptage, 1988) in the active sites. There is only one crystal structure in the PDB from the IlvD/EDD family (PDB entry 2gp4; Southeast Catalytic reactions of (a) RlArDHT and (b) CcXyDHT.…”