Capture of an intermediate in the catalytic cycle of l -aspartate-β-semialdehyde dehydrogenase

Abstract

The structural analysis of an enzymatic reaction intermediate affords a unique opportunity to study a catalytic mechanism in extraordinary detail. Here we present the structure of a tetrahedral intermediate in the catalytic cycle of aspartate-␤-semialdehyde dehydrogenase (ASADH) from Haemophilus influenzae at 2.0-Å resolution. ASADH is not found in humans, yet its catalytic activity is required for the biosynthesis of essential amino acids in plants and microorganisms. Diaminopimelic acid, also formed by this enzymatic pathway, is an integral component of bacterial cell walls, thus making ASADH an attractive target for the development of new antibiotics. This enzyme is able to capture the substrates aspartate-␤-semialdehyde and phosphate as an active complex that does not complete the catalytic cycle in the absence of NADP. A distinctive binding pocket in which the hemithioacetal oxygen of the bound substrate is stabilized by interaction with a backbone amide group dictates the R stereochemistry of the tetrahedral intermediate. This pocket, reminiscent of the oxyanion hole found in serine proteases, is completed through hydrogen bonding to the bound phosphate substrate.

Structural Basis for a Bispecific NADP+ and CoA Binding Site in an Archaeal Malonyl-Coenzyme A Reductase

“…In complete agreement with the structure of the ASD-aspartyl thioacyl adduct (22,31), the malonyl group is mainly anchored to the polypeptide via its carboxylate group by forming hydrogen bonds with the strictly conserved residues Gln 180 , Arg 241 , and His 248 (Fig. 4).…”

Examination of Key Intermediates in the Catalytic Cycle of Aspartate-β-semialdehyde Dehydrogenase from a Gram-positive Infectious Bacteria

“…These branches were initially identified and partitioned through sequence alignments, and with representative high resolution structures now available from each branch (10,(12)(13)(14), they have now been compared by structural alignments. spASADH is the first member of the Gram-positive bacterial ASADH branch that has been structurally characterized.…”

“…We had previously trapped the tetrahedral hemithioacetal intermediate by soaking the products ASA and phosphate into apo hiASADH crystals (14). Covalent adducts with ASA were obtained in the absence and presence of bound phosphate that provided detailed insights into the first steps of ASADH catalysis.…”

“…The enzymes from these organisms show high sequence and structural homology, whereas a recently determined archaeal ASADH structure (13) contains significant differences in its overall structure and in its coenzyme binding specificity. Insights into the mechanism of ASADH were obtained through the structural characterization of a tetrahedral intermediate in the catalytic cycle of the enzyme from Hemophilus influenzae (14); however, the structure of the putative acyl-enzyme derived from this intermediate has remained elusive.…”

Isolation and Characterization of a Novel Lysine Racemase from a Soil Metagenomic Library

“…The C-terminal portion of Lyr contained a region essential for NAGPR activity including the LIAVPGCYPTAAQL ALKP domain (residues 335 to 352) as a catalytic region (3,13), the GxxGxxG motifs (residues 195 to 201 and 372 to 377) as an NADP-binding region (24), and the ATAHEVSHDL region (residues 266 to 275) for Zn binding (24), which might explain the truncated protein Lyr 188-393 , with about 50% of the specific activity of the native NAGPR enzyme from E. coli. Nevertheless, these two truncated proteins lack racemization activity toward lysine.…”