Modulation of Substrate Specificities of d-Sialic Acid Aldolase through Single Mutations of Val-251

Abstract

In a recent directed-evolution study, Escherichia coli D-sialic acid aldolase was converted by introducing eight point mutations into a new enzyme with relaxed specificity, denoted RSaldolase (also known formerly as L-3-deoxy-manno-2-octulosonic acid (L-KDO) aldolase), which showed a preferred selectivity toward L-KDO. To investigate the underlying molecular basis, we determined the crystal structures of D-sialic acid aldolase and RS-aldolase. All mutations are away from the catalytic center, except for V251I, which is near the opening of the (␣/␤) 8 -barrel and proximal to the Schiff base-forming Lys-165. The change of specificity from D-sialic acid to RS-aldolase can be attributed mainly to the V251I substitution, which creates a narrower sugar-binding pocket, but without altering the chirality in the reaction center. The crystal structures of D-sialic acid aldolase⅐L-arabinose and RS-aldolase⅐hydroxypyruvate complexes and five mutants (V251I, V251L, V251R, V251W, and V251I/V265I) of the D-sialic acid aldolase were also determined, revealing the location of substrate molecules and how the contour of the active site pocket was shaped. Interestingly, by mutating Val251 alone, the enzyme can accept substrates of varying size in the aldolase reactions and still retain stereoselectivity. The engineered D-sialic acid aldolase may find applications in synthesizing unnatural sugars of C 6 to C 10 for the design of antagonists and inhibitors of glycoenzymes.Carbohydrates on cell surfaces play a pivotal role in the molecular recognition processes in various cellular interactions. Interfering with the recognition processes between pathogens and hosts may present therapeutic strategies for infectious diseases (1, 2). D-Sialic acids are widely found in nature, from bacteria to plants and animal tissues. Cell-surface glycoproteins and glycolipids frequently contain D-sialic acids at the termini of the oligosaccharide chains. The invading bacteria and viruses also target the cell-surface sialic acids of their hosts, which constitutes an important step for infectivity (3). On the other hand, the monosaccharide D-3-deoxy-manno-2-octulosonic acid (D-KDO) 2 has never been identified in vertebrates, but it is an important component in Gram-negative bacteria (4 -6). Because D-KDO is vital to the structural stability of bacterial outer membrane, targeting D-KDO and the related biosynthetic enzymes may be a new strategy for future antibacterial agent discovery.Currently, unnatural enantiomeric compounds become increasingly important in pharmaceutical implementations. Enantiomers (D-form) of natural peptides have been used to target cell-surface D-sugars. These D-peptide cell-surface sugar binders might serve as new drug candidates for infectious disease (7) and as tools for studying the interactions between proteins and carbohydrate. Owing to the resistance to hydrolases and high binding affinity to sugars, the unnatural D-peptides can become potential therapeutic agents. Through mirror-image phage display, natural L-peptides can be...

First Functional and Mutational Analysis of Group 3 N-Acetylneuraminate Lyases from Lactobacillus antri and Lactobacillus sakei 23K

“…Similar results as those described in Table 1 were described for EcNAL [32]. Interestingly, the relative activity for D-arabinose was equal to that of EcNAL and LpNAL (Table 1), opening up the possibility of using LaNAL as biocatalyst for the production of 3-deoxy-D-manno-2-octulosonic acid (D-KDO), an important synthon for antimicrobials towards the enzymatic Gram- cell wall assembly [30]. In fact, when LaNAL (1 mg mL −1 ) was used at 37°C in 20 mM phosphate buffer pH 7.0 with a 2∶1 pyruvate:D-arabinose ratio (1.2 M vs 0.6 M), 80% conversion into KDO was obtained in 120 hours (Figure 3).…”

“…However, the effect on kinetic parameters of P193Y mutant could be related with a reduction in size of the active site pocket and the conformational changes induced in the neighboring residue E195, which is directly involved in the binding of the O8 and O9 atoms of Neu5Ac. In addition, the relatively good KDO synthetic activity of LaNAL could also be explained by the relatively small sugar-binding pocket, as previously described for Val251 EcNAL mutants [30]. In this respect, double mutant G210/Y213G was also able to convert five carbons sugars (D-mannose and D-arabinose) in the aldol condensation reaction into their corresponding eight carbon acids [2-keto-3-deoxy-d-glycero-galactononulosonic (KDN) and KDO], but a slower rate (up to ∼10% and ∼1% in seven days, respectively).…”

“…The second mutant (G210S) analyzed the first glycine in the aldol-cleavage block, which is highly conserved in all phylogenetic groups, except in group 4.1, where it was replaced by a serine [13]. The NAL crystal structures [8], [10], [30], [31], [33] confirmed the conservation of this glycine because any other residue would restrict substrate binding. In fact, the in silico LaNAL model showed that this mutation clashes with Y213 (data not shown).…”

Cloning, expression, purification, crystallization and preliminary X-ray diffraction studies ofN-acetylneuraminate lyase from methicillin-resistantStaphylococcus aureus

“…An X-ray diffraction data set to a resolution of 1.70 Å was collected from this crystal. This resolution is comparable to that of the wild-type unliganded structure of Nacetylneuraminate lyase from H. influenzae (1.60 Å ; PDB entry 1f6k; Barbosa et al, 2000) and the wild-type unliganded structure of Nacetylneuraminate lyase from E. coli (1.48 Å ; PDB entry 3lbm; Chou et al, 2011).…”

Enzyme‐Catalyzed Aldol Additions

“…The change of specificity from d-NeuA to l-NeuA can be attributed mainly to the Val251Ile substitution, which creates a narrower sugar-binding pocket, and l-Kdo can be better accommodated. By mutating Val25Ile alone, the enzyme can accept substrates of varying size in the aldolase reactions and still retain stereoselectivity [42].…”