The tetrameric assembly of 2-aminomuconic 6-semialdehyde dehydrogenase is a functional requirement of cofactor NAD+ binding.

The bacterium Pseudomonas sp. AP-3 is able to use the environmental pollutant 2-aminophenol as its sole source of carbon, nitrogen, and energy. Eight genes (amnA, B, C, D, E, F, G, and H) encoding 2-aminophenol metabolizing enzymes are clustered into a single operon. 2-Aminomuconic 6-semialdehyde dehydrogenase (AmnC), a member of the aldehyde dehydrogenase (ALDH) superfamily, is responsible for oxidizing 2-aminomuconic 6-semialdehyde to 2-aminomuconate. In contrast to many other members of the ALDH superfamily, the structural basis of the catalytic activity of AmnC remains elusive. Here, we present the crystal structure of AmnC, which displays a homotetrameric quaternary assembly that is directly involved in its enzymatic activity. The tetrameric state of AmnC in solution was also presented using small-angle X-ray scattering. The tetramerization of AmnC is mediated by the assembly of a protruding hydrophobic beta-strand motif and residues V121 and S123 located in the NAD+ -binding domain of each subunit. Dimeric mutants of AmnC dramatically lose NAD+ binding affinity and failed to oxidize the substrate analogue 2-hydroxymuconate-6-semialdehyde to α-hydroxymuconic acid, indicating that tetrameric assembly of AmnC is functional requirement.

Crystallographic and spectroscopic snapshots reveal a dehydrogenase in action.

Aldehydes are ubiquitous intermediates in metabolic pathways and their innate reactivity can often make them quite unstable. There are several aldehydic intermediates in the metabolic pathway for tryptophan degradation that can decay into neuroactive compounds that have been associated with numerous neurological diseases. An enzyme of this pathway, 2-aminomuconate-6-semialdehyde dehydrogenase, is responsible for 'disarming' the final aldehydic intermediate. Here we show the crystal structures of a bacterial analogue enzyme in five catalytically relevant forms: resting state, one binary and two ternary complexes, and a covalent, thioacyl intermediate. We also report the crystal structures of a tetrahedral, thiohemiacetal intermediate, a thioacyl intermediate and an NAD(+)-bound complex from an active site mutant. These covalent intermediates are characterized by single-crystal and solution-state electronic absorption spectroscopy. The crystal structures reveal that the substrate undergoes an E/Z isomerization at the enzyme active site before an sp(3)-to-sp(2) transition during enzyme-mediated oxidation.

Complete nucleotide sequence and functional analysis of the genes for 2-aminophenol metabolism from Pseudomonas sp. AP-3.

A 13.9-kb region, which contained the 2-aminophenol 1,6-dioxygenase genes (amnBA) reported before, was cloned from the 2-aminophenol-assimilating bacterium Pseudomonas sp. AP-3. The complete nucleotide sequence of this region was determined and six genes were found downstream of amnBA. The eight genes together were designated amnBACFEDHG. Each gene was similar to the corresponding gene operating in the meta-cleavage pathway, except for amnB, amnA, and amnD. The four 2-aminophenol-metabolizing enzymes, 2-aminomuconic 6-semialdehyde dehydrogenase, 2-aminomuconate deaminase, 4-oxalocrotonate decarboxylase, and 2-oxopent-4-enoate hydratase, were purified and characterized. NH2-terminal amino acid sequences of each purified enzyme agreed with those deduced from amnC, amnF, amnE, and amnD, respectively. These genes were therefore assigned as the genes encoding these respective proteins. The tight clustering of the amn genes, which were all transcribed in the same direction, raised the possibility that these genes formed a single operon. The organization of the amn genes was entirely different from that of the atd, dmp, and xyl genes reported in the meta-cleavage pathway, although these latter genes clustered similarly.

Purification, characterization, and sequence analysis of 2-aminomuconic 6-semialdehyde dehydrogenase from Pseudomonas pseudoalcaligenes JS45.

2-Aminonumconic 6-semialdehyde is an unstable intermediate in the biodegradation of nitrobenzene and 2-aminophenol by Pseudomonas pseudoalcaligenes JS45. Previous work has shown that enzymes in cell extracts convert 2-aminophenol to 2-aminomuconate in the presence of NAD+. In the present work, 2-aminomuconic semialdehyde dehydrogenase was purified and characterized. The purified enzyme migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 57 kDa. The molecular mass of the native enzyme was estimated to be 160 kDa by gel filtration chromatography. The optimal pH for the enzyme activity was 7.3. The enzyme is able to oxidize several aldehyde analogs, including 2-hydroxymuconic semialdehyde, hexaldehyde, and benzaldehyde. The gene encoding 2-aminomuconic semialdehyde dehydrogenase was identified by matching the deduced N-terminal amino acid sequence of the gene with the first 21 amino acids of the purified protein. Multiple sequence alignment of various semialdehyde dehydrogenase protein sequences indicates that 2-aminomuconic 6-semialdehyde dehydrogenase has a high degree of identity with 2-hydroxymuconic 6-semialdehyde dehydrogenases.

Studies of the catabolic pathway of degradation of nitrobenzene by Pseudomonas pseudoalcaligenes JS45: removal of the amino group from 2-aminomuconic semialdehyde.

Pseudomonas pseudoalcaligenes JS45 utilizes nitrobenzene as the sole source of nitrogen, carbon, and energy. Previous studies have shown that degradation of nitrobenzene involves the reduction of nitrobenzene to nitrosobenzene and hydroxylaminobenzene, followed by rearrangement to 2-aminophenol, which then undergoes meta ring cleavage to 2-aminomuconic semialdehyde. In the present paper, we report the enzymatic reactions responsible for the release of ammonia after ring cleavage. 2-Aminomuconic semialdehyde was oxidized to 2-aminomuconate in the presence of NAD by enzymes in crude extracts. 2-Aminomuconate was subsequently deaminated stoichiometrically to 4-oxalocrotonic acid. No cofactors are required for the deamination. Two enzymes, 2-aminomuconic semialdehyde dehydrogenase and a novel 2-aminomuconate deaminase, distinguished by partial purification of the crude extracts, catalyzed the two reactions. 4-Oxalocrotonic acid was further degraded to pyruvate and acetaldehyde. The key enzyme, 2-aminomuconate deaminase, catalyzed the hydrolytic deamination that released ammonia, which served as the nitrogen source for growth of the organism.