A Reciprocal Single Mutation Affects the Metal Requirement of 3-Deoxy-d-manno-2-octulosonate-8-phosphate (KDO8P) Synthases from Aquifex pyrophilus and Escherichia coli

Abstract

The enzyme 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthase is metal-dependent in one class of organisms and metal-independent in another. We have used a rapid transient kinetic approach combined with site-directed mutagenesis to characterize the role of the metal ion as well as to explore the catalytic mechanisms of the two classes of enzymes. In the metal-dependent Aquifex pyrophilus KDO8P synthase, Cys 11 was replaced by Asn (ApC11N), and in the metal-independent Escherichia coli KDO8P synthase a reciprocal mutation, Asn 26 to Cys, was prepared (EcN26C). The ApC11N mutant retained about 10% of the wild-type maximal activity in the absence of metal ions. Addition of divalent metal ions did not affect the catalytic activity of the mutant enzyme and its catalytic efficiency (k cat /K m ) was reduced by only ϳ12-fold, implying that the ApC11N KDO8P synthase mutant has become a bone fide metal-independent enzyme. The isolated EcN26C mutant had similar metal content and spectral properties as the metal-dependent wild-type A. pyrophilus KDO8P synthase. EDTA-treated EcN26C retained about 6% of the wild-type activity, and the addition of Mn 2؉ or Cd 2؉ stimulated its activity to ϳ30% of the wild-type maximal activity. This suggests that EcN26C KDO8P synthase mutant has properties similar to that of metal-dependent KDO8P synthases. The combined data indicate that the metal ion is not directly involved in the chemistry of the KDO8P synthase catalyzed reaction, but has an important structural role in metal-dependent enzymes in maintaining the correct orientation of the substrates and/or reaction intermediate(s) in the enzyme active site.The enzyme 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) 1 synthase (EC 4.1.2.16) catalyzes the condensation reaction between D-arabinose-5-phosphate (A5P) and phosphoenolpyruvate (PEP) to form KDO8P and inorganic phosphate (P i ) (see Scheme 1) (1). This enzymatic reaction plays an essential role in the assembly process of lipopolysaccharides of most Gram-negative bacteria, and therefore represents an attractive target for the design of novel antibacterial drugs (2, 3). While earlier studies on Escherichia coli KDO8P synthase have established that this enzyme does not require metals (1), it has recently been demonstrated that enzymes from the hyperthermophilic bacteria Aquifex aeolicus (4), Aquifex pyrophilus (5), and from the pathogenic bacterium Helicobacter pylori (6), require a divalent metal cofactor for catalysis. Furthermore, phylogenetic analysis (7, 8) suggests that KDO8P synthases from other pathogenic bacteria, i.e. Chlamydia trachomatis, Chlamydia pneumoniae, and Campylobacter jejuni, may also be metal-dependent enzymes. This is reminiscent of another class of enzymes, aldolases, which catalyze a similar aldol-type C-C bond formation (9). Class I aldolases, which are primarily found in animals and higher plants, do not require a metal cofactor for catalysis. In contrast, Class II aldolases, found predominantly in prokaryotes, use a divalent metal cofactor that...

Characterization of a Metal-independent CAZy Family 6 Glycosyltransferase from Bacteroides ovatus

“…These results show that the catalytic properties of BoGT6a and metal-dependent GT6s are closely similar. Although this may be contrary to expectation, it is not unique; for example, bacterial 3-deoxy-D-manno-octulosonate 8-phosphate synthases can be interconverted between obligate metal dependence and metal independence by substitution of Asn for a metal-binding Cys (38,39).…”

Identification and Characterization of NeuB3 from Campylobacter jejuni as a Pseudaminic Acid Synthase

“…18 O-Enriched water (95%) was purchased from Icon Isotopes. 2-Amino-6-mercapto-7-methylpurine ribonucleoside was purchased from Berry and Associates.…”

“…The extent of 18 O incorporation at the C-2 position was determined to be 54% by mass spectrometry: 16 O, 85). 31 P NMR spectroscopy was used to determine that the position of the label was at the C-2 position:…”

“…2). In each case, catalysis resulted in the production of 18 O-labeled inorganic phosphate indicating that a C-O bond cleavage process was occurring (15,19,21).…”

“…Formation of the tetrahedral intermediate was proposed to proceed in a stepwise fashion through an oxocarbenium ion intermediate. Two other well studied PEP condensing enzymes that utilize very similar mechanisms are 2-keto-3-deoxy-D-manno-octulosonate-8-phosphate synthase, which catalyzes the condensation of PEP and D-arabino-5-phosphate (17,18), and 2-keto-3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, which catalyzes the condensation of PEP and D-erythrose-4-phosphate (19,20). A key mechanistic experiment performed with all three of these synthases was the use of [2-18 O]PEP as a substrate during enzymatic incubations (see labeled atoms in Fig.…”