The short-chain oxidoreductase Q9HYA2 from Pseudomonas aeruginosa PAO1 contains an atypical catalytic center

Abstract

The characteristic oxidation or reduction reaction mechanisms of short-chain oxidoreductase (SCOR) enzymes involve a highly conserved Asp-Ser-Tyr-Lys catalytic tetrad. The SCOR enzyme Q9HYA2 from the pathogenic bacterium Pseudomonas aeruginosa was recognized to possess an atypical catalytic tetrad composed of Lys118-Ser146-Thr159-Arg163. Orthologs of Q9HYA2 containing the unusual catalytic tetrad along with conserved substrate and cofactor recognition residues were identified in 27 additional species, the majority of which are bacterial pathogens. However, this atypical catalytic tetrad was not represented within the Protein Data Bank. The crystal structures of unligated and NADPH-complexed Q9HYA2 were determined at 2.3 A resolution. Structural alignment to a polyketide ketoreductase (KR), a typical SCOR, demonstrated that Q9HYA2's Lys118, Ser146, and Arg163 superimposed upon the KR's catalytic Asp114, Ser144, and Lys161, respectively. However, only the backbone of Q9HYA2's Thr159 overlapped KR's catalytic Tyr157. The Thr159 hydroxyl in apo Q9HYA2 is poorly positioned for participating in catalysis. In the Q9HYA2-NADPH complex, the Thr159 side chain was modeled in two alternate rotamers, one of which is positioned to interact with other members of the tetrad and the bound cofactor. A chloride ion is bound at the position normally occupied by the catalytic tyrosine hydroxyl. The putative active site of Q9HYA2 contains a chemical moiety at each catalytically important position of a typical SCOR enzyme. This is the first observation of a SCOR protein with this alternate catalytic center that includes threonine replacing the catalytic tyrosine and an ion replacing the hydroxyl moiety of the catalytic tyrosine.

Figure 1

Pseudomonas Q9HYA2 structure. A: A surface representation of the holo Q9HYA2 tetramer as present within the asymmetric unit. Each monomer is labeled a–d and individually colored. B: Sequence alignment of the catalytic center of a SCOR enzyme (polyketide ketoreductase, PDB: 1X7H) structurally homologous to Q9HYA2. Catalytic residues of interest have been highlighted with triangles. 1X7H is a typical SCOR enzyme containing the conserved Asn-Ser-Tyr-Lys catalytic tetrad. Q9HYA2 contains a Lys-Ser-Thr-Arg tetrad. The complete alignment is in Supporting Information Figure 3. C: Stereoview of the superposition of apo Q9HYA2 (monomer B; teal) and holo Q9HYA2 (monomer C; green) with NADPH, Arg40, secondary structure elements, and N and C-termini labeled. Regions displaying the greatest differences caused by NADPH binding are displayed in orange on the holo structure. D: Stereoview of superposition of the canonical Asn111-Ser144-Tyr157-Lys161 SCOR catalytic tetrad (1X7H monomer A; teal) with the corresponding residues (Lys118-Ser146-Thr159-Arg163) in holo Q9HYA2 (monomer A; green). Thr159 has two alternate side chain conformations. An interactive view is available in the electronic version of the article.PRO384 Figure 1