Structural determinants in bacterial 2‐keto‐3‐deoxy‐D‐gluconate dehydrogenase KduD for dual‐coenzyme specificity

Abstract

Short-chain dehydrogenase/reductase (SDR) is distributed in many organisms, from bacteria to humans, and has significant roles in metabolism of carbohydrates, lipids, amino acids, and other biomolecules. An important intermediate in acidic polysaccharide metabolism is 2-keto-3-deoxy-d-gluconate (KDG). Recently, two short and long loops in Sphingomonas KDG-producing SDR enzymes (NADPH-dependent A1-R and NADH-dependent A1-R') involved in alginate metabolism were shown to be crucial for NADPH or NADH coenzyme specificity. Two SDR family enzymes-KduD from Pectobacterium carotovorum (PcaKduD) and DhuD from Streptococcus pyogenes (SpyDhuD)-prefer NADH as coenzyme, although only PcaKduD can utilize both NADPH and NADH. Both enzymes reduce 2,5-diketo-3-deoxy-d-gluconate to produce KDG. Tertiary and quaternary structures of SpyDhuD and PcaKduD and its complex with NADH were determined at high resolution (approximately 1.6 Å) by X-ray crystallography. Both PcaKduD and SpyDhuD consist of a three-layered structure, α/β/α, with a coenzyme-binding site in the Rossmann fold; similar to enzymes A1-R and A1-R', both arrange the two short and long loops close to the coenzyme-binding site. The primary structures of the two loops in PcaKduD and SpyDhuD were similar to those in A1-R' but not A1-R. Charge neutrality and moderate space at the binding site of the nucleoside ribose 2' coenzyme region were determined to be structurally crucial for dual-coenzyme specificity in PcaKduD by structural comparison of the NADH- and NADPH-specific SDR enzymes. The corresponding site in SpyDhuD was negatively charged and spatially shallow. This is the first reported study on structural determinants in SDR family KduD related to dual-coenzyme specificity. Proteins 2016; 84:934-947. © 2016 Wiley Periodicals, Inc.

4-Deoxy-l-erythro-5-hexoseulose Uronate (DEH) and DEH Reductase: Key Molecule and Enzyme for the Metabolism and Utilization of Alginate

“…In addition, the molecular surface charge calculated at pH 7.0 shows that the corresponding site is positively charged in A1-R, but slightly negative in A1-R', and this charge may be crucial for the binding or repulsion of the negatively charged NADPH phosphate. These differences in space and charge are caused by two loops, which are referred to as the short and the long loops (A1-R, short loop 13 TGSSQ 17 , long loop 37 HGRKAPA 43 ; A1-R', short loop 13 TGSTE 17 , long loop 37 NSHVDPA 43 ), and the structural features in the two loops are probably important for coenzyme specificity (Figure 6).…”

“…Molecules 2022, 27, x FOR PEER REVIEW 10 of 14 charged NADPH phosphate. These differences in space and charge are caused by two loops, which are referred to as the short and the long loops (A1-R, short loop 13 TGSSQ 17 , long loop 37 HGRKAPA 43 ; A1-R', short loop 13 TGSTE 17 , long loop 37 NSHVDPA 43 ), and the structural features in the two loops are probably important for coenzyme specificity (Figure 6). Adaptively evolved yeast (S. cerevisiae) transformed with the A1-R' gene produced the mutant A1-R' with a Glu-to-Gly substitution at position 17 [9] as described below.…”

“…The short and long loops are conserved in SDR superfamily enzymes, and structural features caused by these loops have been found to be determinants of coenzyme specificity in two enzymes in this superfamily, KduD (Figure 5B) and DhuD (Figure 5C), for the metabolism of pectin and glycosaminoglycan, respectively [43]. Pectobacterium carotovorum KduD has an uncharged coenzyme-binding site that is intermediate in size between A1-R and A1-R', and this enzyme uses both NADH and NADPH as a coenzyme.…”

“…α-helix, brown; β-strand, blue; loop and turn; gray; NADP + , yellow. (B) NAD + -bound KduD[43]. α-helix, purple; β-strand, blue; loop and turn; dark yellow; NADP + , yellow.…”

Enhanced propagation of Granulicatella adiacens from human oral microbiota by hyaluronan

“…For example, streptococci distributed in various tissues, such as the oral, gut, and urogenital organs, are known to degrade and/or assimilate HA 19 , 20 , and streptococcal HA lyases release unsaturated HA disaccharides from HA 21 . Previous reports have indicated that unsaturated HA disaccharides are incorporated into bacterial cells by phosphotransferase system (PTS) 22 and degraded and metabolized to glyceraldehyde-3-phosphate and pyruvate by cytoplasmic enzymes 23 – 25 , such as unsaturated glucuronyl hydrolase (UGL), 4-deoxy- l - threo -5-hexosulose-uronate ketol-isomerase (DhuI), 2-keto-3-deoxy- d -gluconate dehydrogenase (DhuD), 2-keto-3-deoxy- d -gluconate kinase (KdgK), and 2-keto-3-deoxy- d -phosphogluconate aldolase (KdgA; Supplementary Fig. 1 a).…”