John Perozich

The first structure of an aldehyde dehydrogenase reveals novel interactions between NAD and the Rossmann fold.

The first structure of an aldehyde dehydrogenase (ALDH) is described at 2.6 Å resolution. Each subunit of the dimeric enzyme contains an NAD-binding domain, a catalytic domain and a bridging domain. At the interface of these domains is a 15 Å long funnel-shaped passage with a 6 × 12 Å opening leading to a putative catalytic pocket. A new mode of NAD binding, which differs substantially from the classic β-α-β binding mode associated with the ‘Rossmann fold’, is observed which we term the β-α,β mode. Sequence comparisons of the class 3 ALDH with other ALDHs indicate a similar polypeptide fold, novel NAD-binding mode and catalytic site for this family. A mechanism for enzymatic specificity and activity is postulated.

Roles of conserved residues in the arginase family.

Arginases and related enzymes metabolize arginine or similar nitrogen-containing compounds to urea or formamide. In the present report a sequence alignment of 31 members of this family was generated. The alignment, together with the crystal structure of rat liver arginase, allowed the assignment of possible functional or structural roles to 32 conserved residues and conservative substitutions. Two of these residues were previously identified as functionally essential by analysis of inherited defects in the type I arginase gene. Nearly half of the conserved residues are either glycines or prolines located at critical bends in the protein structure. Most metal-coordinating residues, including one histidine and four aspartic acid residues, are strictly conserved. Two additional histidines involved in metal-binding and catalysis are conserved in all arginases and in almost all other family members. Two positions with invariant similarities may serve as indirect metal ligands. Evolutionary relationships within this family were also suggested. Vertebrate type I and II arginases appear to have developed independently from an early gene duplication event. A ureohydrolase sequence from Caenorhabditis elegans is more closely related to other arginases than previously appreciated, while unclassified enzymes from Methanococcus jannaschii and Methanothermus fervidus appear more similar to arginase-related enzymes. In addition, enzymes from Arabidopsis thaliana and Synechocystis, previously identified as arginases, more closely resemble arginase-related enzymes than currently known arginases.

The Big Book of Aldehyde Dehydrogenase Sequences

Traditionally, research on aldehyde dehydrogenases (ALDHs, EC 1.2.1) has focused on three groups: class 1, 2, and 3 ALDHs. Class 1 and 2 ALDHs are very closely related homotetramers and both participate in ethanol metabolism, though class 1 ALDHs can oxidize wide range of metabolites, including retinal (reviewed Lindahl, 1982; Yoshida et al., 1). Class 3 ALDHs appear at first glance to be highly divergent from the other 2 classes. Class 3 ALDHs share only about 25% sequence identity with class 1 and 2 ALDHs and are homodimers. However, in main-chain folding, the structures of the class 2 and 3 monomers are nearly identical (Hempel et al., 1999)

Aldehyde Dehydrogenase Catalytic Mechanism

Elsewhere in this volume we detail findings from an alignment of 145 ALDH sequences (Perozich et al., 1999), and previously at these meetings we reported that the crystal structure of a class 3 ALDH (E-NAD binary complex) revealed a non-traditional mode of NAD-binding within an open β/α domain otherwise familiar in the NAD-binding “ossmann folds” of other dehydrogenases (Liu et al., 1997a). The variability of residues in the substrate-binding site clearly indicates evolutionary tailoring of the substrate specificities of inddual ALDHs. However, farther to the interior of the active site-between the catalytic thiol and NAD molecule where hydride transfer from aldehyde to NAD occurs-strict conservations are compatible with a common chemical mechanism (Liu et al., 1997b)he position of NAD in an isomorphous class 3 ALDH derivative and the emergence of Asn4/169 as a strictly conserved residue prompted us to consider the catalytic mechanism we present here.

UDP-Glucose Dehydrogenase

UDP-glucose dehydrogenase (UDPGDH; EC 1.1.1.22) belongs to a small family of NAD+-linked oxidoreductases which transfer four electrons per catalytic cycle. Other examples of four-electron-transferring enzymes include histidinol dehydrogenase, β-hy-droxy-s-methylglutaryl-CoA reductase, and other nucleotide sugar dehydrogenases (Feingold and Franzen, 1981). The bovine liver enzyme is an apparent homohexamer of 52kDa subunits which appears to function as a trimer of dimers due to its half-sites reactivity to iodoacetate and iodoacetamide (Franzen et al., 1980). We have recently completed the first primary structure of UDPGDH from a mammalian source (bovine liver) (Hempel et al., 1994). Here we present additional details of the analysis and results of a search for potentially homologous proteins using profile analysis.

An algorithm for identification and ranking of family-specific residues, applied to the ALDH3 family

An algorithm for detecting amino acid residues characteristic of individual protein families from within aligned collections of paralogous sequences, and its application to the ALDH3 family versus the rest of the ALDH extended family is described. Residues illuminated by this analysis include a key intramolecular tether, a lysine that makes an intersubunit contact at the dimer interface, three residues in close association with the substrate-binding funnel, and a pair of residues suggested to participate in proton relay during the catalytic cycle.

Beyond the catalytic core of ALDH: a web of important residues begins to emerge

Site-directed mutagenesis was performed in class 3 aldehyde dehydrogenase (ALDH) on both strictly conserved, non-glycine residues, Glu-333 and Phe-335. Both lie in Motif 8 and are indicated to be of central catalytic importance from their positions in the tertiary structure. In addition, a highly conserved residue at the end of Motif 8, Pro-337, and Asp-247, which interacts with the main chain of Motif 8, were also mutated. All substitutions were conservative. Kinetic values clearly show that Glu-333 and Phe-335 are crucial to efficient catalysis, along with Asp-247. Pro-337 appears to have a different role, most likely relating to folding.

Conserved Residues in the Aldehyde Dehydrogenase Family

As described in the preceding chapter (Liu et al., 1996), features of the class 3 ALDH Rossmann fold were totally unexpected. While Gly-187 is involved in binding NAD, its role is completely different from that observed for the first glycine residue of the GXGXXG motif in other dehydrogenases with traditional Rossmann folds. It was thus of interest, once the sequence was fit into the electron density, to examine the locations of strictly conserved residues from our earlier multiple sequence alignment (Hempel et al., 1993). A total of 23 residues were strictly conserved in that alignment. This chapter will confine itself to an attempt to correlate the role of the strictly conserved residues in the ALDH family with the class 3 ALDH (binary complex with NAD) tertiary structure.

Analysis of nucleotide diphosphate sugar dehydrogenases reveals family and group-specific relationships

UDP‐glucose dehydrogenase (UDPGDH), UDP‐N‐acetyl‐mannosamine dehydrogenase (UDPNAMDH) and GDP‐mannose dehydrogenase (GDPMDH) belong to a family of NAD+‐linked 4‐electron‐transfering oxidoreductases called nucleotide diphosphate sugar dehydrogenases (NDP‐SDHs). UDPGDH is an enzyme responsible for converting UDP‐d‐glucose to UDP‐d‐glucuronic acid, a product that has different roles depending on the organism in which it is found. UDPNAMDH and GDPMDH convert UDP‐N‐acetyl‐mannosamine to UDP‐N‐acetyl‐mannosaminuronic acid and GDP‐mannose to GDP‐mannuronic acid, respectively, by a similar mechanism to UDPGDH. Their products are used as essential building blocks for the exopolysaccharides found in organisms like Pseudomonas aeruginosa and Staphylococcus aureus. Few studies have investigated the relationships between these enzymes. This study reveals the relationships between the three enzymes by analysing 229 amino acid sequences. Eighteen invariant and several other highly conserved residues were identified, each serving critical roles in maintaining enzyme structure, coenzyme binding or catalytic function. Also, 10 conserved motifs that included most of the conserved residues were identified and their roles proposed. A phylogenetic tree demonstrated relationships between each group and verified group assignment. Finally, group entropy analysis identified novel conservations unique to each NDP‐SDH group, including residue positions critical to NDP‐sugar substrate interaction, enzyme structure and intersubunit contact. These positions may serve as targets for future research.

In silico analysis of heme oxygenase structural homologues identifies group‐specific conservations

Heme oxygenases (HO) catalyze the breakdown of heme, aiding the recycling of its components. Several other enzymes have homologous tertiary structures to HOs, while sharing little sequence homology. These homologues include thiaminases, the hydroxylase component of methane monooxygenases, and the R2 component of Class I ribonucleotide reductases (RNR). This study compared these structural homologues of HO, using a large number of protein sequences for each homologue. Alignment of a total of 472 sequences showed little sequence conservation, with no residues having conservation in more than 80% of aligned sequences and only five residues conserved in at least 60% of the sequences. Fourteen additional positions, most of which were critical for hydrophobic packing, displayed amino acid similarity of 60% or higher. Ten conserved sequence motifs were identified in HOs and RNRs. Phylogenetic analysis verified the existence of the four distinct groups of HO homologues, which were then analyzed by group entropy analysis to identify residues critical to the unique function of each enzyme. Other methods for determining functional residues were also performed. Several common index positions identified represent critical evolutionary changes that resulted in the unique function of each enzyme, suggesting potential targets for site‐directed mutagenesis. These positions included residues that coordinate ligands, form the active sites, and maintain enzyme structure.