PDBsum entry 6bwc

Biosynthetic protein

PDB id: 6bwc

Contents

Protein chains

(+ 0 more) 324 a.a.

Ligands

NAP ×6

UD1 ×6

SO4 ×5

Waters ×134

PDB id:

6bwc

Name:

Biosynthetic protein

Title:

X-ray structure of pen from bacillus thuringiensis

Structure:

Polysaccharide biosynthesis protein capd. Chain: a, b, c, d, e, f. Engineered: yes. Mutation: yes

Source:

Bacillus thuringiensis hd-771. Organism_taxid: 1218175. Gene: btg_02000. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

2.70Å R-factor: 0.204 R-free: 0.251

Authors:

N.A.Delvaux,J.B.Thoden,H.M.Holden

Key ref:

N.A.Delvaux et al. (2018). Molecular architectures of Pen and Pal: Key enzymes required for CMP-pseudaminic acid biosynthesis in Bacillus thuringiensis. Protein Sci, 27, 738-749. PubMed id: 29266550

Date:

14-Dec-17 Release date: 17-Jan-18

Protein chains

J3UJH9  (J3UJH9_BACTU) -

Protein Sci 27:738-749 (2018)

PubMed id: 29266550

Molecular architectures of Pen and Pal: Key enzymes required for CMP-pseudaminic acid biosynthesis in Bacillus thuringiensis.

N.A.Delvaux, J.B.Thoden, H.M.Holden.

ABSTRACT

Bacillus thuringiensis is a soil-dwelling Gram positive bacterium that has been utilized as a biopesticide for well over 60 years. It is known to contain flagella that are important for motility. One of the proteins found in flagella is flagellin, which is post-translationally modified by O-glycosylation with derivatives of pseudaminic acid. The biosynthetic pathway for the production of CMP-pseudaminic acid in B. thuringiensis, starting with UDP-N-acetyl-d-glucosamine (UDP-GlcNAc), requires seven enzymes. Here, we report the three-dimensional structures of Pen and Pal, which catalyze the first and second steps, respectively. Pen contains a tightly bound NADP(H) cofactor whereas Pal is isolated with bound NAD(H). For the X-ray analysis of Pen, the site-directed D128N/K129A mutant variant was prepared in order to trap its substrate, UDP-GlcNAc, into the active site. Pen adopts a hexameric quaternary structure with each subunit showing the bilobal architecture observed for members of the short-chain dehydrogenase/reductase superfamily. The hexameric quaternary structure is atypical for most members of the superfamily. The structure of Pal was determined in the presence of UDP. Pal adopts the more typical dimeric quaternary structure. Taken together, Pen and Pal catalyze the conversion of UDP-GlcNAc to UDP-4-keto-6-deoxy-l-N-acetylaltrosamine. Strikingly, in Gram negative bacteria such as Campylobacter jejuni and Helicobacter pylori, only a single enzyme (FlaA1) is required for the production of UDP-4-keto-6-deoxy-l-N-acetylaltrosamine. A comparison of Pen and Pal with FlaA1 reveals differences that may explain why FlaA1 is a bifunctional enzyme whereas Pen and Pal catalyze the individual steps leading to the formation of the UDP-sugar product. This investigation represents the first structural analysis of the enzymes in B. thuringiensis that are required for CMP-pseudaminic acid formation.