Zhibing Lu

The X-ray Crystal Structures of Human {alpha}-Phosphomannomutase 1 Reveal the Structural Basis of Congenital Disorder of Glycosylation Type 1a

Congential disorder of glycosylation type 1a (CDG-1a) is a congenital disease characterized by severe defects in nervous system development. It is caused by mutations in α-phosphomannomutase (of which there are two isozymes, α-PMM1 and α-PPM2). Here we report the x-ray crystal structures of human α-PMM1 in the open conformation, with and without the bound substrate, α-d-mannose 1-phosphate. α-PMM1, like most haloalkanoic acid dehalogenase superfamily (HADSF) members, consists of two domains, the cap and core, which open to bind substrate and then close to provide a solvent-exclusive environment for catalysis. The substrate phosphate group is observed at a positively charged site of the cap domain, rather than at the core domain phosphoryl-transfer site defined by the Asp19 nucleophile and Mg2+ cofactor. This suggests that substrate binds first to the cap and then is swept into the active site upon cap closure. The orientation of the acid/base residue Asp21 suggests that α-phosphomannomutase (α-PMM) uses a different method of protecting the aspartylphosphate from hydrolysis than the HADSF member β-phosphoglucomutase. It is hypothesized that the electrostatic repulsion of positive charges at the interface of the cap and core domains stabilizes α-PMM1 in the open conformation and that the negatively charged substrate binds to the cap, thereby facilitating its closure over the core domain. The two isozymes, α-PMM1 and α-PMM2, are shown to have a conserved active-site structure and to display similar kinetic properties. Analysis of the known mutation sites in the context of the structures reveals the genotype-phenotype relationship underlying CDG-1a.

The catalytic scaffold of the haloalkanoic acid dehalogenase enzyme superfamily acts as a mold for the trigonal bipyramidal transition state

The evolution of new catalytic activities and specificities within an enzyme superfamily requires the exploration of sequence space for adaptation to a new substrate with retention of those elements required to stabilize key intermediates/transition states. Here, we propose that core residues in the large enzyme family, the haloalkanoic acid dehalogenase enzyme superfamily (HADSF) form a “mold” in which the trigonal bipyramidal transition states formed during phosphoryl transfer are stabilized by electrostatic forces. The vanadate complex of the hexose phosphate phosphatase BT4131 from Bacteroides thetaiotaomicron VPI-5482 (HPP) determined at 1.00 Å resolution via X-ray crystallography assumes a trigonal bipyramidal coordination geometry with the nucleophilic Asp-8 and one oxygen ligand at the apical position. Remarkably, the tungstate in the complex determined to 1.03 Å resolution assumes the same coordination geometry. The contribution of the general acid/base residue Asp-10 in the stabilization of the trigonal bipyramidal species via hydrogen-bond formation with the apical oxygen atom is evidenced by the 1.52 Å structure of the D10A mutant bound to vanadate. This structure shows a collapse of the trigonal bipyramidal geometry with displacement of the water molecule formerly occupying the apical position. Furthermore, the 1.07 Å resolution structure of the D10A mutant complexed with tungstate shows the tungstate to be in a typical “phosphate-like” tetrahedral configuration. The analysis of 12 liganded HADSF structures deposited in the protein data bank (PDB) identified stringently conserved elements that stabilize the trigonal bipyramidal transition states by engaging in favorable electrostatic interactions with the axial and equatorial atoms of the transferring phosphoryl group.

Structure-Function Analysis of 2-Keto-3-deoxy-D-glycero-D-galactonononate-9-phosphate Phosphatase Defines Specificity Elements in Type C0 Haloalkanoate Dehalogenase Family Members

The phosphotransferases of the haloalkanoate dehalogenase superfamily (HADSF) act upon a wide range of metabolites in all eukaryotes and prokaryotes and thus constitute a significant force in cell function. The challenge posed for biochemical function assignment of HADSF members is the identification of the structural determinants that target a specific metabolite. The “8KDOP” subfamily of the HADSF is defined by the known structure and catalytic activity of 2-keto-3-deoxy-8-phospho-d-manno-octulosonic acid (KDO-8-P) phosphatase. Homologues of this enzyme have been uniformly annotated as KDO-8-P phosphatase. One such gene, BT1713, from the Bacteroides thetaiotaomicron genome was recently found to encode the enzyme 2-keto-3-deoxy-d-glycero-d-galacto-9-phosphonononic acid (KDN-9-P) phosphatase in the biosynthetic pathway of the 9-carbon α-keto acid, 2-keto-3-deoxy-d-glycero-d-galactonononic acid (KDN). To find the structural elements that provide substrate-specific interactions and to allow identification of genomic sequence markers, the x-ray crystal structures of BT1713 liganded to the cofactor Mg2+and complexed with tungstate or \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{VO_{3}^{-}}\) \end{document}/Neu5Ac were determined to 1.1, 1.85, and 1.63 Å resolution, respectively. The structures define the active site to be at the subunit interface and, as confirmed by steady-state kinetics and site-directed mutagenesis, reveal Arg-64*, Lys-67*, and Glu-56 to be the key residues involved in sugar binding that are essential for BT1713 catalytic function. Bioinformatic analyses of the differentially conserved residues between BT1713 and KDO-8-P phosphatase homologues guided by the knowledge of the structure-based specificity determinants define Glu-56 and Lys-67* to be the key residues that can be used in future annotations.

A Structural Element that Facilitates Proton-Coupled Electron Transfer in Oxalate Decarboxylase

The conformational properties of an active-site loop segment, defined by residues Ser(161)-Glu(162)-Asn(163)-Ser(164), have been shown to be important for modulating the intrinsic reactivity of Mn(II) in the active site of Bacillus subtilis oxalate decarboxylase. We now detail the functional and structural consequences of removing a conserved Arg/Thr hydrogen-bonding interaction by site-specific mutagenesis. Hence, substitution of Thr-165 by a valine residue gives an OxDC variant (T165V) that exhibits impaired catalytic activity. Heavy-atom isotope effect measurements, in combination with the X-ray crystal structure of the T165V OxDC variant, demonstrate that the conserved Arg/Thr hydrogen bond is important for correctly locating the side chain of Glu-162, which mediates a proton-coupled electron transfer (PCET) step prior to decarboxylation in the catalytically competent form of OxDC. In addition, we show that the T165V OxDC variant exhibits a lower level of oxalate consumption per dioxygen molecule, consistent with the predictions of recent spin-trapping experiments [Imaram et al. (2011) Free Radicals Biol. Med. 50, 1009-1015]. This finding implies that dioxygen might participate as a reversible electron sink in two putative PCET steps and is not merely used to generate a protein-based radical or oxidized metal center.

The X-ray crystallographic structure and specificity profile of HAD superfamily phosphohydrolase BT1666: Comparison of paralogous functions in B. thetaiotaomicron.

Analysis of the haloalkanoate dehalogenase superfamily (HADSF) has uncovered homologues occurring within the same organism that are found to possess broad, overlapping substrate specificities, and low catalytic efficiencies. Here we compare the HADSF phosphatase BT1666 from Bacteroides thetaiotaomicron VPI‐5482 to a homologue with high sequence identity (40%) from the same organism BT4131, a known hexose‐phosphate phosphatase. The goal is to find whether these enzymes represent duplicated versus paralogous activities. The X‐ray crystal structure of BT1666 was determined to 1.82 Å resolution. Superposition of the BT1666 and BT4131 structures revealed a conserved fold and identical active sites suggestive of a common physiological substrate. The steady‐state kinetic constants for BT1666 were determined for a diverse panel of phosphorylated metabolites to define its substrate specificity profile and overall level of catalytic efficiency. Whereas BT1666 and BT4131 are both promiscuous, their substrate specificity profiles are distinct. The catalytic efficiency of BT1666 (kcat/Km = 4.4 × 102M−1 s−1 for the best substrate fructose 1,6‐(bis)phosphate) is an order of magnitude less than that of BT4131 (kcat/Km = 6.7 × 103M−1 s−1 for 2‐deoxyglucose 6‐phosphate). The seemingly identical active‐site structures point to sequence variation outside the active site causing differences in conformational dynamics or subtle catalytic positioning effects that drive the divergence in catalytic efficiency and selectivity. The overlapping substrate profiles may be understood in terms of differential regulation of expression of the two enzymes or a conferred advantage in metabolic housekeeping functions by having a larger range of possible metabolites as substrates. Proteins 2011;.

Human symbiont Bacteroides thetaiotaomicron synthesizes 2-keto-3-deoxy-D-glycero-D- galacto-nononic acid (KDN).

The proper functioning of the human intestine is dependent on its bacterial symbionts, the most predominant of which belong to the Phylum Bacteroidetes. These bacteria are known to use variable displays of multiple capsular polysaccharides (CPs) to aid in their survival and foraging within the intestine. Bacteroides thetaiotaomicron is a prominent human gut symbiont and a remarkably versatile glycophile. The structure determination of the CPs, encoded by the eight CP loci, is the key to understanding the mechanism of this organisms adaptation on a molecular level. Herein, we report the bioinformatics-based discovery and chemical demonstration of a biosynthetic pathway that forms and cytidylates 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN), most likely for inclusion in the CP encoded by B. thetaiotaomicron CP locus 7.