| Annotation aspect | Functional annotation summary | Supporting citations |
|---|---|---|
| Identity | UniProt Q88DU2 is DnaK, the canonical bacterial Hsp70 chaperone, in *Pseudomonas putida* KT2440; it was directly identified as DnaK (Q88DU2) in a KT2440 proteomics study. | (pqac-00000002) |
| Domains/architecture | DnaK/Hsp70 has the expected bacterial architecture: N-terminal ~45 kDa nucleotide-binding domain (NBD), substrate-binding β-domain (SBDβ), α-helical lid (SBDα), and a short intrinsically disordered C-terminal tail; ATP binding occurs at the NBD and peptide substrates bind the SBD. | (pqac-00000005, pqac-00000015) |
| Mechanism | DnaK is an ATP-dependent molecular chaperone that binds and releases non-native polypeptides through allosterically coupled ATP hydrolysis cycles; ATP-state DnaK has low substrate affinity, whereas after ATP hydrolysis substrate affinity rises ~10–50-fold and association/dissociation rates drop ~100–1000-fold. | (pqac-00000006, pqac-00000015) |
| Co-chaperones | The core bacterial KJE system comprises DnaK with the J-domain cochaperone DnaJ and nucleotide-exchange factor GrpE; DnaJ stimulates Hsp70 ATPase activity by >1,000-fold, and GrpE promotes ADP release and substrate release. | (pqac-00000004, pqac-00000005, pqac-00000015) |
| Localization | The primary functional localization is cytosolic, consistent with proteostasis and folding roles; in KT2440, DnaK (Q88DU2) was also detected on the surface of isolated PHA granules/carbonosomes under nitrogen-limiting PHA-producing conditions, though authors note this may reflect true association or preparation carryover. | (pqac-00000002, pqac-00000020, pqac-00000022) |
| Pathways/biological processes | DnaK participates in the bacterial heat-shock/protein quality-control network, assists co- and post-translational folding, helps prevent aggregation/remodel damaged proteins, and is connected to nutrient-stress responses such as nitrogen limitation/PHA accumulation and osmotic stress adaptation. | (pqac-00000014, pqac-00000015, pqac-00000020, pqac-00000022) |
| Phenotypes in *P. putida* | In *P. putida*, DnaK appears essential because deletion attempts failed; its disordered C-terminus and especially a negatively charged terminal motif contribute to competitive fitness and facilitate toxicity of the GraT toxin. Overexpression increased dnaK mRNA ~2.7-fold, and C-terminal motif mutants were outcompeted in long-term competition, especially at 34 °C. | (pqac-00000012, pqac-00000013, pqac-00000015) |
| Recent 2024 structural/regulatory insights | Recent 2024 work sharpened DnaK annotation beyond classical folding: bacterial DnaK is present in 98.9% of bacterial genomes, and 6.4% encode ≥2 paralogs; cryo-EM of DnaK–GrpE showed an asymmetric 1:2 complex where GrpE ratchets to couple ADP release and substrate release; a 2024 stress study showed DnaK can also reduce translation independently of J-domain cochaperones under specific stress. | (pqac-00000005, pqac-00000006, pqac-00000004, pqac-00000007) |
| Applications/implementations | DnaK-related knowledge is being leveraged for biotechnology and stress engineering: KT2440 salt-tolerance engineering identified dnaK/dnaJ/clpB/htpG among stress-responsive chaperones; engineered KT2440 strains tolerated up to 5% NaCl, and up to 6% with compatible solutes, enabling aromatic degradation under 4% NaCl. In broader bacterial engineering, chaperone systems are used to improve robustness under industrial stresses. | (pqac-00000018, pqac-00000019, pqac-00000014) |
| Key quantitative data | Representative quantitative findings relevant to annotation: 98.9% of bacterial genomes encode dnaK and 6.4% have multiple paralogs; ATP hydrolysis shifts DnaK substrate affinity by ~10–50-fold; under Mg2+ starvation DnaK-ribosome association increased ~3-fold while Trigger Factor association fell ~30-fold; C-terminal DnaK truncation caused ~10,000-fold viability loss in that stress model; KT2440 salt-tolerance engineering enabled 56.70% benzoic acid and 95.64% protocatechuic acid degradation at 4% NaCl in 48 h. | (pqac-00000005, pqac-00000006, pqac-00000004, pqac-00000007, pqac-00000018, pqac-00000019) |


*Table: This table summarizes the most relevant identity, mechanistic, localization, pathway, phenotype, and application evidence for *Pseudomonas putida* KT2440 DnaK (UniProt Q88DU2). It is designed as a compact annotation aid with direct citation IDs for each major claim.*