Structural and Biochemical Investigation of PglF from <i>Campylobacter jejuni</i> Reveals a New Mechanism for a Member of the Short Chain Dehydrogenase/Reductase Superfamily

Riegert, A.S.; Thoden, James B.; Schoenhofen, Ian C.; Watson, David; Young, N. Martin; Tipton, Peter A.; Holden, Hazel M.

doi:10.1021/acs.biochem.7b00910

Cited by 24 publications

(31 citation statements)

References 45 publications

(78 reference statements)

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“…We also showed that the insertion of the pen-pal and pglF genes into the amyE locus of the Δ spsM mutant strain restored Leg production. Pen-Pal and PglF were previously shown to convert UDP-GlcNAc to UDP-4-keto-6-deoxy-GlcNAc ( 43 , 49 ). More surprisingly, the addition of the legB gene in the Δ spsM mutant strain did not fully restore Leg production.…”

Section: Discussionmentioning

confidence: 99%

The sps Genes Encode an Original Legionaminic Acid Pathway Required for Crust Assembly in Bacillus subtilis

Dubois

Krzewinski

Yamakawa

et al. 2020

mBio

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The crust is the outermost spore layer of most Bacillus strains devoid of an exosporium. This outermost layer, composed of both proteins and carbohydrates, plays a major role in the adhesion and spreading of spores into the environment. Recent studies have identified several crust proteins and have provided insights about their organization at the spore surface. However, although carbohydrates are known to participate in adhesion, little is known about their composition, structure, and localization. In this study, we showed that the spore surface of Bacillus subtilis is covered with legionaminic acid (Leg), a nine-carbon backbone nonulosonic acid known to decorate the flagellin of the human pathogens Helicobacter pylori and Campylobacter jejuni. We demonstrated that the spsC, spsD, spsE, spsG, and spsM genes of Bacillus subtilis are required for Leg biosynthesis during sporulation, while the spsF gene is required for Leg transfer from the mother cell to the surface of the forespore. We also characterized the activity of SpsM and highlighted an original Leg biosynthesis pathway in B. subtilis. Finally, we demonstrated that Leg is required for the assembly of the crust around the spores, and we showed that in the absence of Leg, spores were more adherent to stainless steel probably because of their reduced hydrophilicity and charge. IMPORTANCE Bacillus species are a major economic and food safety concern of the food industry because of their food spoilage-causing capability and persistence. Their persistence is mainly due to their ability to form highly resistant spores adhering to the surfaces of industrial equipment. Spores of the Bacillus subtilis group are surrounded by the crust, a superficial layer which plays a key role in their adhesion properties. However, knowledge of the composition and structure of this layer remains incomplete. Here, for the first time, we identified a nonulosonic acid (Leg) at the surfaces of bacterial spores (B. subtilis). We uncovered a novel Leg biosynthesis pathway, and we demonstrated that Leg is required for proper crust assembly. This work contributes to the description of the structure and composition of Bacillus spores which has been under way for decades, and it provides keys to understanding the importance of carbohydrates in Bacillus adhesion and persistence in the food industry.

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Section: Discussionmentioning

confidence: 99%

The sps Genes Encode an Original Legionaminic Acid Pathway Required for Crust Assembly in Bacillus subtilis

Dubois

Krzewinski

Yamakawa

et al. 2020

mBio

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“…The final step is the transfer of the proton from the conserved glutamate back to C‐5′ and transfer of the hydride from NADH to C‐6′ to yield the final product. A recent report has shown that the tyrosine residue is not strictly conserved, however, and a different catalytic mechanism has been put forth which does not invoke the use of a tyrosinate base …”

Section: Discussionmentioning

confidence: 99%

Biochemical analysis of a sugar 4,6‐dehydratase from Acanthamoeba polyphaga Mimivirus

2020

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The exciting discovery of the giant DNA Mimivirus in 2003 challenged the conventional description of viruses in a radical way, and since then, dozens of additional giant viruses have been identified. It has now been demonstrated that the Mimivirus genome encodes for the two enzymes required for the production of the unusual sugar 4-amino-4,6-dideoxy-D-glucose, namely a 4,6-dehydratase and an aminotransferase. In light of our long-standing interest in the bacterial 4,6-dehydratases and in unusual sugars in general, we conducted a combined structural and functional analysis of the Mimivirus 4,6-dehydratase referred to as R141. For this investigation, the threedimensional X-ray structure of R141 was determined to 2.05 Å resolution and refined to an R-factor of 18.3%. The overall fold of R141 places it into the short-chain dehydrogenase/reductase (SDR) superfamily of proteins. Whereas its molecular architecture is similar to that observed for the bacterial 4,6-dehydratases, there are two key regions where the polypeptide chain adopts different conformations. In particular, the conserved tyrosine that has been implicated as a catalytic acid or base in SDR superfamily members is splayed away from the active site by nearly 12 Å, thereby suggesting that a major conformational change must occur upon substrate binding. In addition to the structural analysis, the kinetic parameters for R141 using either dTDP-D-glucose or UDP-D-glucose as substrates were determined. Contrary to a previous report, R141 demonstrates nearly identical catalytic efficiency with either nucleotide-linked sugar. The data presented herein represent the first threedimensional model for a viral 4,6-dehydratase and thus expands our understanding of these fascinating enzymes. K E Y W O R D SAcanthamoeba polyphaga Mimivirus, dideoxysugar, giant viruses, UDP-D-glucose 4,6-dehydratase, UDP-D-viosamine, viral glycans, X-ray structure Abbreviations: dTDP, thymidine diphosphate; ESI, electrospray ionization; HEPES, N-2-hydroxyethylpiperazine-N 0 -2-ethanesulfonic acid; HEPPS, N-2-hydroxyethylpiperazine-N 0 -3-propanesulfonic acid; HPLC, high-performance liquid chromatography; NAD + , nicotinamide adenine dinucleotide (oxidized); NADH, nicotinamide adenine dinucleotide (reduced); NADP + , nicotinamide adenine dinucleotide phosphate (oxidized); NADPH, nicotinamide adenine dinucleotide phosphate (reduced); Ni-NTA, nickel-nitrilotriacetic acid; PCR, polymerase chain reaction; PLP, pyridoxal 5 0 -phosphate; Qui4N, 4-amino-4,6-dideoxy-D-glucose; TEV, Tobacco Etch Virus; Tris, tris-(hydroxymethyl)aminomethane; UDP, uridine diphosphate.

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“…A buffered solution of 50 mM UDP-GlcNAc was prepared in 50 mM HEPPS (pH 8.0). To this solution, 20 mg/ml of PglF, a UDP- N -acetyl-glucosamine 4,6-dehydratase from C. jejuni , was added ( 8 ). The reaction was allowed to proceed overnight at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Characterization of two enzymes from Psychrobacter cryohalolentis that are required for the biosynthesis of an unusual diacetamido-d-sugar

Linehan

Thoden

Holden

2021

Journal of Biological Chemistry

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Psychrobacter cryohalolentis strain K5 T is a Gram-negative organism first isolated in 2006. It has a complex O-antigen that contains, in addition to l -rhamnose and d -galactose, two diacetamido- and a triacetamido-sugar. The biochemical pathways for the production of these unusual sugars are presently unknown. Utilizing the published genome sequence of the organism, we hypothesized that the genes 0612, 0638, and 0637 encode for a 4,6-dehydratase, an aminotransferase, and an N -acetyltransferase, respectively, which would be required for the biosynthesis of one of the diacetamido-sugars, 2,4-diacetamido-2,4,6-trideoxy- d -glucose, starting from UDP- N -acetylglucosamine. Here we present functional and structural data on the proteins encoded by the 0638 and 0637 genes. The kinetic properties of these enzymes were investigated by a discontinuous HPLC assay. An X-ray crystallographic structure of 0638, determined in its external aldimine form to 1.3 Å resolution, demonstrated the manner in which the UDP ligand is positioned into the active site. It is strikingly different from that previously observed for PglE from Campylobacter jejuni , which functions on the same substrate. Four X-ray crystallographic structures were also determined for 0637 in various complexed states at resolutions between 1.3 and 1.55 Å. Remarkably, a tetrahedral intermediate mimicking the presumed transition state was trapped in one of the complexes. The data presented herein confirm the hypothesized functions of these enzymes and provide new insight into an unusual sugar biosynthetic pathway in Gram-negative bacteria. We also describe an efficient method for acetyl-CoA synthesis that allowed us to overcome its prohibitive cost for this analysis.

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Structural and Biochemical Investigation of PglF from Campylobacter jejuni Reveals a New Mechanism for a Member of the Short Chain Dehydrogenase/Reductase Superfamily

Cited by 24 publications

References 45 publications

The sps Genes Encode an Original Legionaminic Acid Pathway Required for Crust Assembly in Bacillus subtilis

The sps Genes Encode an Original Legionaminic Acid Pathway Required for Crust Assembly in Bacillus subtilis

Biochemical analysis of a sugar 4,6‐dehydratase from Acanthamoeba polyphaga Mimivirus

Characterization of two enzymes from Psychrobacter cryohalolentis that are required for the biosynthesis of an unusual diacetamido-d-sugar

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