Hui-Min Qin

Structure and Polymannuronate Specificity of a Eukaryotic Member of Polysaccharide Lyase Family 14.

Alginate is an abundant algal polysaccharide, composed of β-d-mannuronate and its C5 epimer α-l-guluronate, that is a useful biomaterial in cell biology and tissue engineering, with applications in cancer and aging research. The alginate lyase (EC 4.2.2.3) from Aplysia kurodai, AkAly30, is a eukaryotic member of the polysaccharide lyase 14 (PL-14) family and degrades alginate by cleaving the glycosidic bond through a β-elimination reaction. Here, we present the structural basis for the substrate specificity, with a preference for polymannuronate, of AkAly30. The crystal structure of AkAly30 at a 1.77 Å resolution and the putative substrate-binding model show that the enzyme adopts a β-jelly roll fold at the core of the structure and that Lys-99, Tyr-140, and Tyr-142 form catalytic residues in the active site. Their arrangements allow the carboxyl group of mannuronate residues at subsite +1 to form ionic bonds with Lys-99. The coupled tyrosine forms a hydrogen bond network with the glycosidic bond, and the hydroxy group of Tyr-140 is located near the C5 atom of the mannuronate residue. These interactions could promote the β-elimination of the mannuronate residue at subsite +1. More interestingly, Gly-118 and the disulfide bond formed by Cys-115 and Cys-124 control the conformation of an active-site loop, which makes the space suitable for substrate entry into subsite −1. The cleavage efficiency of AkAly30 is enhanced relative to that of mutants lacking either Gly-118 or the Cys-115–Cys-124 disulfide bond. The putative binding model and mutagenesis studies provide a novel substrate recognition mode explaining the polymannuronate specificity of PL-14 alginate lyases.

Crystal structure of a novel N-substituted L-amino acid dioxygenase from Burkholderia ambifaria AMMD.

A novel dioxygenase from Burkholderia ambifaria AMMD (SadA) stereoselectively catalyzes the C3-hydroxylation of N-substituted branched-chain or aromatic L-amino acids, especially N-succinyl-L-leucine, coupled with the conversion of α-ketoglutarate to succinate and CO2. To elucidate the structural basis of the substrate specificity and stereoselective hydroxylation, we determined the crystal structures of the SadA.Zn(II) and SadA.Zn(II).α-KG complexes at 1.77 Å and 1.98 Å resolutions, respectively. SadA adopted a double-stranded β-helix fold at the core of the structure. In addition, an HXD/EXnH motif in the active site coordinated a Zn(II) as a substitute for Fe(II). The α-KG molecule also coordinated Zn(II) in a bidentate manner via its 1-carboxylate and 2-oxo groups. Based on the SadA.Zn(II).α-KG structure and mutation analyses, we constructed substrate-binding models with N-succinyl-L-leucine and N-succinyl-L-phenylalanine, which provided new insight into the substrate specificity. The results will be useful for the rational design of SadA variants aimed at the recognition of various N-succinyl L-amino acids.

Structural optimization of SadA, an Fe(II)- and α-ketoglutarate-dependent dioxygenase targeting biocatalytic synthesis of N-succinyl-L-threo-3,4-dimethoxyphenylserine.

L-threo-3,4-Dihydroxyphenylserine (l-DOPS, Droxidopa) is a psychoactive drug and synthetic amino acid precursor that acts as a prodrug to the neurotransmitters. SadA, a dioxygenase from Burkholderia ambifaria AMMD, is an Fe(II)- and α-ketoglutarate (KG)-dependent enzyme that catalyzes N-substituted branched-chain or aromatic l-amino acids. SadA is able to produce N-succinyl-l-threo-3,4-dimethoxyphenylserine (NSDOPS), which is a precursor of l-DOPS, by catalyzing the hydroxylation of N-succinyl-3,4-dimethoxyphenylalanine (NSDOPA). However, the catalytic activity of SadA toward NSDOPS is much lower than that toward N-succinyl branched-chain l-amino acids. Here, we report an improved biocatalytic synthesis of NSDOPS with SadA. Structure-based protein engineering was applied to improve the α-KG turnover activity for the synthesis of NSDOPS. The G79A, G79A/F261W or G79A/F261R mutant showed a more than 6-fold increase in activity compared to that of the wild-type enzyme. The results provide a new insight into the substrate specificity toward NSDOPA and will be useful for the rational design of SadA mutants as a target of industrial biocatalysts.

Crystal structure of conjugated polyketone reductase (CPR-C1) from Candida parapsilosis IFO 0708 complexed with NADPH

Conjugated polyketone reductase (CPR‐C1) from Candida parapsilosis IFO 0708 is a member of the aldo–keto reductase (AKR) superfamily and reduces ketopantoyl lactone to d‐pantoyl lactone in a NADPH‐dependent and stereospecific manner. We determined the crystal structure of CPR‐C1.NADPH complex at 2.20 Å resolution. CPR‐C1 adopted a triose‐phosphate isomerase (TIM) barrel fold at the core of the structure in which Thr25 and Lys26 of the GXGTX motif bind uniquely to the adenosine 2′‐phosphate group of NADPH. This finding provides a novel structural basis for NADPH binding of the AKR superfamily. Proteins 2013; 81:2059–2063.

Expression, purification, crystallization and preliminary X-ray analysis of a novel N-substituted branched-chain L-amino-acid dioxygenase from Burkholderia ambifaria AMMD.

Ferrous ion- and α-ketoglutarate-dependent dioxygenase from Burkholderia ambifaria AMMD (SadA) catalyzes the C3-hydroxylation of N-substituted branched-chain L-amino acids, especially N-succinyl-L-leucine, coupled to the conversion of α-ketoglutarate to succinate and CO(2). SadA was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method at 293 K. Crystals of selenomethionine-substituted SadA were obtained using a reservoir solution containing PEG 3000 as the precipitant at pH 9.5 and diffracted X-rays to 2.4 Å resolution. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 49.3, b = 70.9, c = 148.2 Å. The calculated Matthews coefficient (V(M) = 2.1 Å(3) Da(-1), 41% solvent content) suggested that the crystal contains two molecules per asymmetric unit.

Laminarinase from Flavobacterium sp. reveals the structural basis of thermostability and substrate specificity

Laminarinase from Flavobacterium sp. strain UMI-01, a new member of the glycosyl hydrolase 16 family of a marine bacterium associated with seaweeds, mainly degrades β-1,3-glucosyl linkages of β-glucan (such as laminarin) through the hydrolysis of glycosidic bonds. We determined the crystal structure of ULam111 at 1.60-Å resolution to understand the structural basis for its thermostability and substrate specificity. A calcium-binding motif located on the opposite side of the β-sheet from catalytic cleft increased its degrading activity and thermostability. The disulfide bridge Cys31-Cys34, located on the β2-β3 loop near the substrate-binding site, is responsible for the thermostability of ULam111. The substrates of β-1,3-linked laminarin and β-1,3-1,4-linked glucan bound to the catalytic cleft in a completely different mode at subsite -3. Asn33 and Trp113, together with Phe212, formed hydrogen bonds with preferred substrates to degrade β-1,3-linked laminarin based on the structural comparisons. Our structural information provides new insights concerning thermostability and substrate recognition that will enable the design of industrial biocatalysts.

Author Correction: Laminarinase from Flavobacterium sp. reveals the structural basis of thermostability and substrate specificity

A correction to this article has been published and is linked from the HTML version of this paper. The error has not been fixed in the paper.

Structure change for substrate recognition in conjugated polyketone reductase

1Department of Applied Biological Chemistry, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. 2Division of Applied Life Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai 559-8531, Japan. 3Division of Applied Life Sciences, Kyoto University, Kitashirakawa-Oiwakecho, Sakyoku, Kyoto 606-8502, Japan. 4Faculty of Bioenvironmental Science, Kyoto Gakuen University, Sogabe-cho, Kameoka 6218555, Japan. E-mail:ahmqin@mail.ecc.u-tokyo.ac.jp