KdgF, the missing link in the microbial metabolism of uronate sugars from pectin and alginate

Abstract

Uronates are charged sugars that form the basis of two abundant sources of biomass-pectin and alginate-found in the cell walls of terrestrial plants and marine algae, respectively. These polysaccharides represent an important source of carbon to those organisms with the machinery to degrade them. The microbial pathways of pectin and alginate metabolism are well studied and essentially parallel; in both cases, unsaturated monouronates are produced and processed into the key metabolite 2-keto-3-deoxygluconate (KDG). The enzymes required to catalyze each step have been identified within pectinolytic and alginolytic microbes; yet the function of a small ORF, kdgF, which cooccurs with the genes for these enzymes, is unknown. Here we show that KdgF catalyzes the conversion of pectin-and alginate-derived 4,5-unsaturated monouronates to linear ketonized forms, a step in uronate metabolism that was previously thought to occur spontaneously. Using enzyme assays, NMR, mutagenesis, and deletion of kdgF, we show that KdgF proteins from both pectinolytic and alginolytic bacteria catalyze the ketonization of unsaturated monouronates and contribute to efficient production of KDG. We also report the X-ray crystal structures of two KdgF proteins and propose a mechanism for catalysis. The discovery of the function of KdgF fills a 50-y-old gap in the knowledge of uronate metabolism. Our findings have implications not only for the understanding of an important metabolic pathway, but also the role of pectinolysis in plant-pathogen virulence and the growing interest in the use of pectin and alginate as feedstocks for biofuel production.uronate | ring opening | tautomerization | pectin | alginate P olysaccharides, such as those found as biomass in marine algae and terrestrial plants, comprise a vast sink of photosynthetically fixed carbon and a potentially enormous source of energy to organisms with the appropriate metabolic systems to unlock it. Pectin, a complex polyuronate largely made up of the uronate D-galacturonate connected by α-(1,4) linkages, is one such polysaccharide. The principal reservoir of pectin is the primary cell wall of terrestrial plants and, as such, its degradation and metabolism by microbes is an important component of the natural turnover of biomass, infection by plant pathogens, digestion of dietary fiber in the mammalian gut, and the utilization of biomass as feedstocks for biofuels or other valuable products (1). Similarly, alginate, another polyuronate that comprises β-linked D-mannuronate and L-guluronate (in varying lengths and arrangements), is a major cell wall component of brown macroalgae (seaweed). Alginate can comprise up to 40% of the dry weight of the algae, making the recycling of this photosynthetically fixed carbon a significant aspect of the ocean carbon cycle. The prevalence of both polysaccharides in biomass has made them important considerations in the production of ethanol from marine algae and terrestrial plant feedstocks (e.g., refs. 2-4).The microbial pathways responsible for the metab...

Aquatic adaptation of a laterally acquired pectin degradation pathway in marine gammaproteobacteria

“…). It is interesting to note that the island encodes a 4‐deoxy‐L‐threo‐5‐hexosulose‐uronate ketol‐isomerase (KduI) and a 2‐deoxy‐D‐gluconate 3‐dehydrogenase (KduD), which have recently been proposed as two enzymes of an alternative pathway for galacturonate degradation in E. coli (Rothe et al ., ), and which can process the unsaturated galacturonate that is produced by GH105 via the kdgF (PH505_dn00060) encoded enzyme (Hobbs et al ., ). Another pectin‐responsive protein of the genomic island is the KDPG aldolase (2‐dehydro‐3‐deoxy‐phosphogluconate aldolase), which catalyzes a late step in the biodegradation of pectin by converting 2‐dehydro‐3‐deoxy‐D‐gluconate 6‐phosphate (KDPG) into pyruvate and D‐glyceraldehyde 3‐phosphate.…”

Biphasic cellular adaptations and ecological implications of Alteromonas macleodii degrading a mixture of algal polysaccharides

“…ED Entner-Doudoroff pathway, IM inner membrane, OM outer membrane Alginate degradation was related to AlgPUL1, which has been characterized in detail before [33]. The enzyme KdgF (Alt831_00946) essential for both pectin and alginate degradation [90] is encoded in AlgPUL1 and hence potentially shared by both pathways. In contrast, both pathways possess a dedicated KdgK enzyme comparable to Saccharophagus degradans [91].…”

A Review of the Human Sigma-1 Receptor Structure

“…The cupin fold is functionally versatile with members including metalloenzymes from several enzyme classes as well as seed storage proteins [20]. Metalloenzymes with the cupin fold have been observed to use Fe, Mn, Ni, Zn, Co, and Cu [20], and include the oxidoreductases thiol dioxygenase [21] and 2-oxoglutarate oxygenase [22], the lyase ectoine synthase, and the hydrolase KdgF [23]. The yeast sterol isomerases that are most similar in sequence to S1R (see below) have a modest affinity to Zn +2 , but S1R does not bind metals with high affinity [24], nor does it exhibit isomerase activity [17].…”