Structure and Function of Cyanobacterial DHDPS and DHDPR

Abstract

Lysine biosynthesis in bacteria and plants commences with a condensation reaction catalysed by dihydrodipicolinate synthase (DHDPS) followed by a reduction reaction catalysed by dihydrodipicolinate reductase (DHDPR). Interestingly, both DHDPS and DHDPR exist as different oligomeric forms in bacteria and plants. DHDPS is primarily a homotetramer in all species, but the architecture of the tetramer differs across kingdoms. DHDPR also exists as a tetramer in bacteria, but has recently been reported to be dimeric in plants. This study aimed to characterise for the first time the structure and function of DHDPS and DHDPR from cyanobacteria, which is an evolutionary important phylum that evolved at the divergence point between bacteria and plants. We cloned, expressed and purified DHDPS and DHDPR from the cyanobacterium Anabaena variabilis. The recombinant enzymes were shown to be folded by circular dichroism spectroscopy, enzymatically active employing the quantitative DHDPS-DHDPR coupled assay, and form tetramers in solution using analytical ultracentrifugation. Crystal structures of DHDPS and DHDPR from A. variabilis were determined at 1.92 Å and 2.83 Å, respectively, and show that both enzymes adopt the canonical bacterial tetrameric architecture. These studies indicate that the quaternary structure of bacterial and plant DHDPS and DHDPR diverged after cyanobacteria evolved.

Defining the distinct, intrinsic properties of the novel type I interferon, IFNϵ

“…Affinity measurements using microscale thermophoresis (MST) were carried out employing a Monolith NT. 115 instrument (NanoTemper Technologies) as previously described (38,39). mIFNAR1-ECD and mIFNAR2-ECDC94S were labeled using the NHS RED NanoTemper labeling kit according to the manufacturer's instructions.…”

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

“…Even when multiple dapA genes have been annotated in a bacterium, such as in Agrobacterium tumefaciens, only one dapA gene has been shown to have bona fide DHDPS activity [36]. These enzymes have similar catalytic efficiencies, which are consistent with other DHDPS orthologues [16,36,51,52]. Alignments of multiple P. aeruginosa strains demonstrate that dapA2, dapA3 and dapA4 are universally present, whereas dapA1 is annotated in only 47% of 2.4 9 10 5 NR k cat /K M ASA (s À1 ÁM À1 ) 2.9 9 10 5 5.5 9 10 5 NR a Data obtained from [24], NR = not reported.…”

“…Recombinant proteins were expressed in E. coli BL21 (DE3) cells as hexa-histidine-tagged constructs and purified using immobilized metal affinity chromatography (IMAC) as previously described [16,63,64]. In brief, the synthetic genes coding for the P. aeruginosa proteins and the PaDHDPS2 mutants H56Q and H56K were purchased from Bioneer (Bioneer Pacific, Kew East, Vic., Australia) ligated in the pET28a expression vector.…”

“…The DAP pathway commences with the condensation of pyruvate and (S)-aspartate semialdehyde (ASA) to form 4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid (HTPA) (Fig. 1) [12,13,15,16]. This first committed and rate-limiting step is catalysed by 4hydroxy-tetrahydrodipicolinate synthase (EC 4.3.3.7), commonly referred to as dihydrodipicolinate synthase (DHDPS) [9,14].…”

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

“…Interestingly, Girish et al () found that DHDPR can also function as a dehydratase in addition to its role as an NAD(P)H‐dependent reductase. The structure of DHDPR has been determined from bacteria (including Gram‐positive; Girish, Navratna, & Gopal, ; Janowski, Kefala, & Weiss, ; Sagong & Kim, ) and Gram‐negative bacteria; Scapin, Reddy, Zheng, & Blanchard, ), cyanobacteria (Christensen et al, ), and plants (Griffin et al, ). These studies have shown that DHDPR is a homotetramer with a unique monomer unit, except for plant‐derived DHDPR.…”