Elucidation of the role of Ser329 and the C-terminal region in the catalytic activity of Pseudomonas stutzeril-rhamnose isomerase

Abstract

Pseudomonas stutzeri l-rhamnose isomerase (l-RhI) is capable of catalyzing the isomerization between various aldoses and ketoses, showing high catalytic activity with broad substrate-specificity compared with Escherichia coli l-RhI. In a previous study, the crystal structure of P. stutzeri l-RhI revealed an active site comparable with that of E. coli l-RhI and d-xylose isomerases (d-XIs) with structurally conserved amino acids, but also with a different residue seemingly responsible for the specificity of P. stutzeri l-RhI, though the residue itself does not interact with the bound substrate. This residue, Ser329, corresponds to Phe336 in E. coli l-RhI and Lys294 in Actinoplanes missouriensis d-XI. To elucidate the role of Ser329 in P. stutzeri l-RhI, we constructed mutants, S329F (E. coli l-RhI type), S329K (A. missouriensis d-XI type), S329L and S329A. Analyses of the catalytic activity and crystal structure of the mutants revealed a hydroxyl group of Ser329 to be crucial for catalytic activity via interaction with a water molecule. In addition, in complexes with substrate, the mutants S329F and S329L exhibited significant electron density in the C-terminal region not observed in the wild-type P. stutzeri l-RhI. The C-terminal region of P. stutzeri l-RhI has flexibility and shows a flip-flop movement at the inter-molecular surface of the dimeric form.

Structure of l‐rhamnose isomerase in complex with l‐rhamnopyranose demonstrates the sugar‐ring opening mechanism and the role of a substrate sub‐binding site

“…The sub‐binding site is expected to recognize the anomer's structure, allowing the β‐anomer to pass into the catalytic site. We previously proposed that the C‐terminal region has a role in the flip‐flop movement on the inter‐molecular surface of Mol‐A/Mol‐B or Mol‐C/Mol‐D, helping substrate‐binding and/or substrate‐release, based on the X‐ray structures and kinetic analysis of mutant P. stutzeri l ‐RhIs [17]. Various conformations of C‐terminal regions were also found in this study.…”

“…The kinetic parameters of enzymes were determined by a cystein–carbazole assay detecting the amount of ketose produced using a calorimetric method. The reaction was initiated by the addition of various concentrations of each substrate (final concentrations of 1, 2, 5, 10, 20, 50, or 100 mM) and terminated by the addition of 50 μl of 10% trichloroacetic acid after the mixture was incubated for 10 min at 50 °C [6,17,34]. Kinetic parameters are listed in Supplemental Table S1.…”

“…The X‐ray structures of l ‐RhIs from E. coli [14] and P. stutzeri [15–17] have been reported previously. l ‐RhI forms a homo tetramer, with two adjacent metal ions at the catalytic site of each subunit.…”

Regulation of the rhaEWRBMA Operon Involved in l -Rhamnose Catabolism through Two Transcriptional Factors, RhaR and CcpA, in Bacillus subtilis

“…After the incubation of rhamnose with His-RhaA, the resultant product was clearly colored by the cysteine-carbazole-sulfuric acid method, indicating that rhamnose was converted to a ketose. Based on the molar extinction coefficient obtained by rhamnulose, the specific activity of HisRhaA for rhamnose was determined to be 58.5 Ϯ 6.2 mol min Ϫ1 mg Ϫ1 (mean value Ϯ the standard deviation in three independent experiments), which was comparable to the value for the rhamnose of Mesorhizobium loti L-rhamnose isomerase (64.5 mol min Ϫ1 mg Ϫ1 ) (41) and somewhat lower than the value for the rhamnose of P. stutzeri L-rhamnose isomerase (280 mol min Ϫ1 mg Ϫ1 ) (38). As shown in Table 3, His-RhaB Ec specifically phosphorylated rhamnulose, and it hardly accepted rhamnose as the substrate (I and II).…”

“…The rhamnose isomerization activity of His-RhaA was measured by a modification of a method described previously (38). The reaction mixture contained, in a total volume of 500 l, 50 mM Tris-Cl (pH 8.0), 1 mM MnCl 2 , 10 mM L-rhamnose (Sigma-Aldrich), and 0.1 to 1 g of the purified His-RhaA.…”