Structure and function of the pyridoxal 5'-phosphate-dependent (PLP) threonine deaminase IlvA1 from Pseudomonas aeruginosa PAO1.

IlvA1, a pyridoxal phosphate-dependent (PLP) enzyme, catalyzes the deamination of l-threonine and l-serine to yield 2-ketobutyric acid or pyruvate. To gain insights into the function of IlvA1, we determined its crystal structure from Pseudomonas aeruginosa to 2.3 Å. Density for a 2-ketobutyric acid product was identified in the active site and a putative allosteric site. Activity and substrate binding assays confirmed that IlvA1 utilizes l-threonine, l-serine, and L-allo-threonine as substrates. The enzymatic activity is regulated by the end products l-isoleucine and l-valine. Additionally, the efficiency of d-cycloserine and l-cycloserine inhibitors on IlvA1 enzymatic activity was examined. Notably, site-directed mutagenesis confirmed the active site residues and revealed that Gln165 enhances the enzyme activity, emphasizing its role in substrate access. This work provides crucial insights into the structure and mechanism of IlvA1 and serves as a starting point for further functional and mechanistic studies of the threonine deaminase in P. aeruginosa.

Structure and mechanism of the γ-glutamyl-γ-aminobutyrate hydrolase SpuA from Pseudomonas aeruginosa.

Polyamines are important regulators in all living organisms and are implicated in essential biological processes including cell growth, differentiation and apoptosis. Pseudomonas aeruginosa possesses an spuABCDEFGHI gene cluster that is involved in the metabolism and uptake of two polyamines: spermidine and putrescine. In the proposed γ-glutamylation-putrescine metabolism pathway, SpuA hydrolyzes γ-glutamyl-γ-aminobutyrate (γ-Glu-GABA) to glutamate and γ-aminobutyric acid (GABA). In this study, crystal structures of P. aeruginosa SpuA are reported, confirming it to be a member of the class I glutamine amidotransferase (GAT) family. Activity and substrate-binding assays confirm that SpuA exhibits a preference for γ-Glu-GABA as a substrate. Structures of an inactive H221N mutant were determined with bound glutamate thioester intermediate or glutamate product, thus delineating the active site and substrate-binding pocket and elucidating the catalytic mechanism. The crystal structure of another bacterial member of the class I GAT family from Mycolicibacterium smegmatis (MsGATase) in complex with glutamine was determined for comparison and reveals a binding site for glutamine. Activity assays confirm that MsGATase has activity for glutamine as a substrate but not for γ-Glu-GABA. The work reported here provides a starting point for further investigation of polyamine metabolism in P. aeruginosa.

Structural characterization of a 2-aminoethylphosphonate:pyruvate aminotransferase from Pseudomonas aeruginosa PAO1.

2-aminoethylphosphonate:pyruvate aminotransferase (AEPT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that mediates the first step in the AEP degradation pathway. It catalyzes the transamination of 2-aminoethylphosphonate (AEP) with pyruvate to phosphonoacetaldehyde and l-alanine respectively. Although the enzyme is widely present in microorganisms, there are few reports on the structure and function of AEPT to date. Here we report the crystal structure of AEPT from Pseudomonas aeruginosa PAO1 (PaAEPT) to 2.35 Å resolution in the absence of the PLP cofactor. PaAEPT crystallizes in space group P21212 with one monomer per asymmetric unit. Analytical ultracentrifugation analysis shows that PaAEPT forms a stable dimer in solution. Our work provides a valuable starting point for further functional and mechanistic studies of the AEP degradation pathway.

Structure of subunit I of the anthranilate synthase complex of Mycolicibacterium smegmatis.

The tryptophan biosynthesis pathway, which does not exist in mammals, is highly conserved in Mycobacterium. Anthranilate synthase (AS) catalyzes the initial reactions in the tryptophan biosynthesis pathway in many microorganisms, catalyzing the conversion of glutamine and chorismate to form pyruvate and anthranilate. Here, the crystal structure of anthranilate synthase component I (AS I) from Mycolicibacterium smegmatis (MsTrpE) has been determined to 1.7 Å resolution. MsTrpE crystallizes in the space group P1 with two monomers in the asymmetric unit, which is consistent with the oligomeric state in solution as confirmed by analytical ultracentrifugation. Inspection of the active site shows that it is in the active form with a bound Mg2+ ion and a ligand that is modelled as benzoate. The position of benzoate mimics the position of the anthranilate product in the active site. The structure of MsTrpE will provide a starting point for the investigation of latent biotechnology and pharmaceutical applications of anthranilate synthase component I.

Structural characterization of the Pseudomonas aeruginosa dehydrogenase AtuB involved in citronellol and geraniol catabolism.

Pseudomonas aeruginosa can metabolize acyclic monoterpenoids (such as citronellol and geraniol) as the only carbon and energy sources. A total of seven proteins (AtuA, AtuB, AtuCF, AtuD, AtuE, AtuG, AtuH) have been identified in Pseudomonas aeruginosa as participating in the acyclic terpene utilization pathway. AtuB is a dehydrogenase enzyme responsible for citronellol and geraniol catabolism in the acyclic terpene utilization (Atu) pathway, although its structure and function have not been characterized to date. Here we report the crystal structure of AtuB from Pseudomonas aeruginosa PAO1 (PaAtuB) to 1.8 Å resolution. PaAtuB crystallizes in the space group F222 with a single monomer in the asymmetric unit. Analytical ultracentrifugation data shows that PaAtuB forms a stable tetramer in solution, which is consistent with the structure. Structural analysis confirms that AtuB belongs to the short-chain dehydrogenase/reductase (SDR) family. AtuB is predicted to bind NADP(H) from the crystal structure, which is confirmed by MicroScale Thermophoresis analysis that shows PaAtuB binds NADP(H) with a Kd value of 258 µM. This work provides a starting point to explore potential biotechnology and pharmaceutical applications of AtuB.

Structural characterization of an acetolactate decarboxylase from Klebsiella pneumoniae.

Acetolactate decarboxylase (ALDC) is a well-characterized anabolic enzyme involved with 3-hydroxy butanone (acetoin), an important physiological metabolite excreted by microbes. Although the enzyme is widely present in microorganisms, few atomic structures and functions of ALDC have been reported to date. Here we report the crystal structure of ALDC from Klebsiella pneumoniae KP (KpALDC). KpALDC crystallizes in space group P3121 with one monomer per asymmetric unit. Analytical ultracentrifugation data shows that KpALDC forms a stable dimer but can exist as a tetramer in solution. A Zn2+ ion is coordinated by three strictly-conserved histidines (His198, His200 and His211) and a conserved glutamate (Glu69), but the C-terminal tail that forms part of the active site in ALDC enzymes is disordered. A complex structure with ethane-1,2-diol shows a unusual mode of binding, whereby the ligand does not coordinate the Zn2+ ion. MicroScale Thermophoresis analysis shows that KpALDC binds Zn2+ ions, but no binding of Mg2+, Ca2+ and Mn2+ ions was detected.