Antenna Domain Mobility and Enzymatic Reaction of l-Rhamnulose-1-phosphate Aldolase^,

Abstract

The enzyme l-rhamnulose-1-phosphate aldolase from Escherichia coli participates in the degradation pathway of l-rhamnose, a ubiquitous deoxy-hexose. It is a homotetramer of the rare C4-symmetric type with N-terminal domains protruding like antennas from the main body. A mobility analysis of the enzyme gave rise to the hypothesis that an anisotropic thermal antenna motion may support the catalysis (Kroemer et al., Biochemistry 42, 10560, 2003). We checked this hypothesis by generating four single mutants and one disulfide bridge that were designed to reduce the mobility of the antenna domain without disturbing the chain-fold or the active center. The catalytic rates of the mutants revealed activity reductions that correlated well with the expected antenna fixation. Among these mutants, K15W was crystallized, structurally elucidated, and used as a guide for modeling the others. The structure confirmed the design because the mutation introduced a tight nonpolar contact to a neighboring subunit that fixed the antenna but did not affect the main chain. The fixation was confirmed by a comparison of the anisotropic B-factors describing the mobility of the domains. It turned out that the distinctly anisotropic mobility of the wild-type antenna domain has become isotropic in K15W, in agreement with the design. We suggest that, like K15W, the other mutations also followed the design, validating the correlation between antenna mobility and activity. This correlation suggests that the domain mobility facilitates the reaction.

Comparative genomics and functional analysis of rhamnose catabolic pathways and regulons in bacteria

“…The L-rhamnulose-1-P aldolase domain in RhaE is distantly homologous to class II aldolases including the analogs enzyme, RhaD, and the L-fuculose-1-P aldolase, FucA, from E. coli . The tertiary structures and catalytic mechanisms for these enzymes have been determined (Dreyer and Schulz, 1996; Grueninger and Schulz, 2008). We aligned the amino acid sequences of all three enzymes using the multiple protein sequence and structure alignment server PROMALS3D (Pei et al, 2008) (Figure S7 in Supplementary Material).…”

Protein Flexibility and Metal Coordination Changes in DHAP‐Dependent Aldolases

“…The N-domain contribution is much larger than all the components of the C-domain. These modes therefore represent rotational motions of the N-domain with respect to the Cdomain, as was captured by the crystallographic ATFs in references [22,23] . The relative contribution of the calculated modes may be different in the crystal, [14,15,44] even so, the agreement of the calculated modes and the ATF in the description of the N-domain rotation is very good.…”

“…The FucA model drawings can be found in Figure S4 in the Supporting Information. For the distance values, see [26] and RhuA, [22,23] respectively. These similar k cat values do not agree with RhuAs energy barrier being sevenfold smaller than FucAs.…”

“…Average ATFs for the N-domain showed a rotational displacement with respect to the C-domain. In a subsequent investigation, [23] mutants were prepared that potentially affected the mobility of these domains but not the active site, and a decrease in the catalytic parameters for RhuA was found. From these results, it was suggested that dynamical movements in RhuA were coupled to the catalytic step.…”

Synthesis of D-Sorbose and D-Psicose by RecombinantEscherichia coli

“…RhaD from E. coli (RhaD E.coli ) is a homotetramer of the rare C4-symmetric type, containing two molecules of Zn 2+ per enzyme complex, and each subunit is composed of 274 amino acid residues with a molecular weight of 30,149 Da [26,27]. Interestingly, Fessner et al first reported in 1991 that RhaD E.coli can also tolerate aldehyde with D-configuration, such as D-GA, as an acceptor, and we discovered in our previous study that Downloaded by [New York University] at 23:35 29 July 2015…”