Escherichia coli dihydrodipicolinate synthase and dihydrodipicolinate reductase: kinetic and inhibition studies of two putative herbicide targets

Coulter, Carolyn V.; Gerrard, Juliet A.; Kraunsoe, James A.E.; Pratt, Andrew J.

doi:10.1002/(sici)1096-9063(199909)55:9<887::aid-ps36>3.3.co;2-2

Cited by 14 publications

(32 citation statements)

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“…The K m of CgDHDPR for NADPH was nearly threefold lower than the equivalent values for NADH (Table 2). However, the K m of EcDHDPR for NADPH was at least fourfold higher than the equivalent values for NADH, which is in agreement with the results of previous studies (Coulter, Gerrard, Kraunsoe, & Pratt, 1999;Scapin et al, 1995). The K cat of CgDHDPR for NADH and NADPH were measured to be 1.9 ± 0.2 and 5.1 ± 0.8 s −1 , respectively, which were in contrast to those of EcDHDPR (K cat = 5.9 ± 0.3 s −1 for NADH, whereas K cat = 2.7 ± 0.4 s −1 for NADPH) ( Table 2).…”

Section: Methodssupporting

confidence: 93%

Rational modification of Corynebacterium glutamicum dihydrodipicolinate reductase to switch the nucleotide‐cofactor specificity for increasing l‐lysine production

Yang

Liu

et al. 2018

Biotech & Bioengineering

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l-lysine is an important amino acid in animals and humans and NADPH is a vital cofactor for maximizing the efficiency of l-lysine fermentation. Dihydrodipicolinate reductase (DHDPR), an NAD(P)H-dependent enzyme, shows a variance in nucleotide-cofactor affinity in bacteria. In this study, we rationally engineered Corynebacterium glutamicum DHDPR (CgDHDPR) to switch its nucleotide-cofactor specificity resulting in an increase in final titer (from 82.6 to 117.3 g L ), carbon yield (from 0.35 to 0.44 g [g glucose] ) and productivity (from 2.07 to 2.93 g L hr ) of l-lysine in JL-6 ΔdapB::Ec-dapB in fed-batch fermentation. To do this, we comparatively analyzed the characteristics of CgDHDPR and Escherichia coli DHDPR (EcDHDPR), indicating that hetero-expression of NADH-dependent EcDHDPR increased l-lysine production. Subsequently, we rationally modified the conserved structure of cofactor-binding motif, and results indicated that introducing the mutation K11A or R13A in CgDHDPR and introducing the mutation R16A or R39A in EcDHDPR modifies the nucleotide-cofactor affinity of DHDPR. Lastly, the effects of these mutated DHDPRs on l-lysine production were investigated. The highest increase (26.2%) in l-lysine production was observed for JL-6 ΔdapB::Ec-dapB , followed by JL-6 Cg-dapB (21.4%) and JL-6 ΔdapB::Ec-dapB (15.2%). This is the first report of a rational modification of DHDPR that enhances the l-lysine production and yield through the modulation of nucleotide-cofactor specificity.

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Section: Methodssupporting

confidence: 93%

Rational modification of Corynebacterium glutamicum dihydrodipicolinate reductase to switch the nucleotide‐cofactor specificity for increasing l‐lysine production

Yang

Liu

et al. 2018

Biotech & Bioengineering

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“…In the first step of the reaction, pyruvate forms a Schiff base with a lysine residue in the active site. This is followed by the binding of the second substrate, (S)-ASA, and subsequent dehydration and cyclization to form HTPA [37]. Structural features of Abbreviations used: (S)-ASA, (S)-aspartate-semialdehyde; DHDPR, dihydrodipicolinate reductase; DHDPS, dihydrodipicolinate synthase; HTPA, (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid; rmsd, root mean square deviation.…”

Section: Introductionmentioning

confidence: 99%

Dihydrodipicolinate synthase from Thermotoga maritima

Pearce

Perugini

McKerchar

et al. 2006

Biochemical Journal

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DHDPS (dihydrodipicolinate synthase) catalyses the branch point in lysine biosynthesis in bacteria and plants and is feedback inhibited by lysine. DHDPS from the thermophilic bacterium Thermotoga maritima shows a high level of heat and chemical stability. When incubated at 90 degrees C or in 8 M urea, the enzyme showed little or no loss of activity, unlike the Escherichia coli enzyme. The active site is very similar to that of the E. coli enzyme, and at mesophilic temperatures the two enzymes have similar kinetic constants. Like other forms of the enzyme, T. maritima DHDPS is a tetramer in solution, with a sedimentation coefficient of 7.2 S and molar mass of 133 kDa. However, the residues involved in the interface between different subunits in the tetramer differ from those of E. coli and include two cysteine residues poised to form a disulfide bond. Thus the increased heat and chemical stability of the T. maritima DHDPS enzyme is, at least in part, explained by an increased number of inter-subunit contacts. Unlike the plant or E. coli enzyme, the thermophilic DHDPS enzyme is not inhibited by (S)-lysine, suggesting that feedback control of the lysine biosynthetic pathway evolved later in the bacterial lineage.

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“…5A). To further validate this, the DHDPS activity of all four gene products was quantitatively assessed using the DHDPS-DHDPR coupled assay [38,41,42]. The dapA1 and dapA2 gene products are shown to have comparable activity to EcDHDPS, whilst the dapA3 and dapA4 gene products have no detectable activity (Fig.…”

Section: Expression Purification and Characterization Of Dapa Gene Pmentioning

confidence: 97%

“…Having determined that the recombinant PaDHDPS1 and PaDHDPS2 enzymes were homogenous, folded and catalytically active, we set out to determine their enzyme kinetic parameters using the DHDPS-DHDPR coupled assay [38,41,46]. Initial rates were monitored while varying the concentrations of the DHDPS substrates, pyruvate and ASA (Fig.…”

Section: Catalytic Efficiency Of Padhdps1 and Padhdps2mentioning

confidence: 99%

“…Dihydrodipicolinate synthase enzyme activity was quantified using the DHDPS-DHDPR coupled assay [37,38,41] that measures substrate turnover spectrophotometrically at Abs 340 nm (e 340 nm = 6220 M À1 Ácm À1 ) via the associated oxidation of NADPH as previously reported. Assays were performed in triplicate (n = 3) at 37°C in a temperaturecontrolled Cary 4000 UV/Vis spectrophotometer (Varian, Mulgrave, Vic., Australia).…”

Section: Enzyme Kineticsmentioning

confidence: 99%

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Identification of two dihydrodipicolinate synthase isoforms from Pseudomonas aeruginosa that differ in allosteric regulation

et al. 2019

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Pseudomonas aeruginosa is one of the leading causes of nosocomial infections, accounting for 10% of all hospital‐acquired infections. Current antibiotics against P. aeruginosa are becoming increasingly ineffective due to the exponential rise in drug resistance. Thus, there is an urgent need to validate and characterize novel drug targets to guide the development of new classes of antibiotics against this pathogen. One such target is the diaminopimelate (DAP) pathway, which is responsible for the biosynthesis of bacterial cell wall and protein building blocks, namely meso‐DAP and lysine. The rate‐limiting step of this pathway is catalysed by the enzyme dihydrodipicolinate synthase (DHDPS), typically encoded for in bacteria by a single dapA gene. Here, we show that P. aeruginosa encodes two functional DHDPS enzymes, PaDHDPS1 and PaDHDPS2. Although these isoforms have similar catalytic activities (kcat = 29 s−1 and 44 s−1 for PaDHDPS1 and PaDHDPS2, respectively), they are differentially allosterically regulated by lysine, with only PaDHDPS2 showing inhibition by the end product of the DAP pathway (IC50 = 130 μm). The differences in allostery are attributed to a single amino acid difference in the allosteric binding pocket at position 56. This is the first example of a bacterium that contains multiple bona fide DHDPS enzymes, which differ in allosteric regulation. We speculate that the presence of the two isoforms allows an increase in the metabolic flux through the DAP pathway when required in this clinically important pathogen. Databases PDB ID: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6P90.

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Escherichia coli dihydrodipicolinate synthase and dihydrodipicolinate reductase: kinetic and inhibition studies of two putative herbicide targets

Cited by 14 publications

References 0 publications

Rational modification of Corynebacterium glutamicum dihydrodipicolinate reductase to switch the nucleotide‐cofactor specificity for increasing l‐lysine production

Rational modification of Corynebacterium glutamicum dihydrodipicolinate reductase to switch the nucleotide‐cofactor specificity for increasing l‐lysine production

Dihydrodipicolinate synthase from Thermotoga maritima

Identification of two dihydrodipicolinate synthase isoforms from Pseudomonas aeruginosa that differ in allosteric regulation

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