Structurally diverse dehydroshikimate dehydratase variants participate in microbial quinate catabolism

Abstract

Quinate and shikimate can be degraded by a number of microbes. Dehydroshikimate dehydratases (DSDs) play a central role in this process, catalyzing the conversion of 3-dehydroshikimate to protocatechuate, a common intermediate of aromatic degradation pathways. DSDs have applications in metabolic engineering for the production of valuable protocatechuate-derived molecules. Although a number of Gram-negative bacteria are known to catabolize quinate and shikimate, only limited information exists on the quinate/shikimate catabolic enzymes found in these organisms. Here, we have functionally and structurally characterized a putative DSD designated QuiC1, which is present in some pseudomonads. The QuiC1 protein is not related by sequence with previously identified DSDs from the Gram-negative genus, Acinetobacter, but instead shows limited sequence identity in its N-terminal half with fungal DSDs. Analysis of a Pseudomonas aeruginosa quiC1 gene knock-out demonstrates that it is important for growth on either quinate or shikimate. The structure of a QuiC1 enzyme from P. putida reveals that the protein is a fusion of two distinct modules: an N-terminal sugar phosphate isomerase-like domain associated with DSD activity and a novel C-terminal hydroxyphenylpyruvate dioxygenase-like domain. The results of this study highlight the considerable diversity of enzymes that participate in quinate/shikimate catabolism in different microbes.

Structural and biochemical approaches uncover multiple evolutionary trajectories of plant quinate dehydrogenases

“…Recombinant protein expression and purification were carried out using an established protocol (Christendat et al ., ; Singh and Christendat, ; Peek et al ., ). Bacterial protein expression plasmids containing the genes for S. lycopersicum QDH, N. tabacum QDH, B. rapa QDH/SDH and B. napus QDH/SDH were transformed into E. coli BL21 CodonPlus (Agilent Technologies, https://www.agilent.com/).…”

“…DHQD activity was assessed using an enzyme‐coupled assay. In this assay, we utilized a microbial dehydroshikimate dehydratase enzyme that converts the dehydroshikimate produced by DHQD to protocatechuate, which can readily be detected by HPLC (Peek et al ., ) (Figure S1). All reaction mixtures were set up with the respective cofactors, NAD + or NADP + , quinate/shikimate and dehydroshikimate dehydratase enzyme.…”

Characterization of theCorynebacterium glutamicumdehydroshikimate dehydratase QsuB and its potential for microbial production of protocatechuic acid

“…This level of identity allowed us to predict the 3D structure of QsuB based on the QuiC1 crystal structure (PDB ID: 5HMQ) (Fig 2). All the residues (Arg64, Leu97, Glu134, Leu136, His168, Asp165, Gln191, Ser206, Arg210 and Glu239) identified previously in the active site of DSD of QuiC1 [14] were also present in the N-terminal domain of QsuB.…”

“…DSD is encoded by the asbF gene and, being identical in Bacillus anthracis and Bacillus thuringiensis , has been thoroughly investigated [12, 13]. Based on primary structure analysis, all known DSDs were divided into four groups: fungal single-domain, bacterial two-domain, AsbF-like, and bacterial membrane-associated enzymes [14]. The application of AsbF and fungal ( Podospora pauciseta ) and bacterial two-domain ( Klebsiella pneumoniae ) enzymes to obtain 3,4-DHBA as an intermediate for the generation of valuable compounds from glucose has been demonstrated [10, 11, 15, 16].…”

“…DSD is encoded by the qsuB gene, a part of the operon qsuABCD , whose products are involved in the quinate/shikimate utilization pathway [17]. Previously, QsuB of C. glutamicum was assigned to the two-domain bacterial DSD group [14]. QuiC1 from Pseudomonas putida was the only characterized member of this class.…”