The Gene glvA of Bacillus subtilis 168 Encodes a Metal-requiring, NAD(H)-dependent 6-Phospho-α-glucosidase

Abstract

The gene glvA (formerly glv-1) from Bacillus subtilis has been cloned and expressed in Escherichia coli. The purified protein GlvA (449 residues, M r ‫؍‬ 50,513) is a unique 6-phosphoryl-O-␣-D-glucopyranosyl:phosphoglucohydrolase (6-phospho-␣-glucosidase) that requires both NAD(H) and divalent metal (Mn 2؉ , Fe 2؉ , Co 2؉ , or Ni 2؉) for activity. 6-Phospho-␣-glucosidase (EC 3.2.1.122) from B. subtilis cross-reacts with polyclonal antibody to maltose 6-phosphate hydrolase from Fusobacterium mortiferum, and the two proteins exhibit amino acid sequence identity of 73%. Estimates for the M r of GlvA determined by SDS-polyacrylamide gel electrophoresis (51,000) and electrospray-mass spectroscopy (50,510) were in excellent agreement with the molecular weight of 50,513 deduced from the amino acid sequence. The sequence of the first 37 residues from the N terminus determined by automated analysis agreed precisely with that predicted by translation of glvA. The chromogenic and fluorogenic substrates, p-nitrophenyl-␣-D-glucopyranoside 6-phosphate and 4-methylumbelliferyl-␣-D-glucopyranoside 6-phosphate were used for the discontinuous assay and in situ detection of enzyme activity, respectively. Site-directed mutagenesis shows that three acidic residues, Asp 41 may function as the catalytic acid (proton donor) and nucleophile (base), respectively, during hydrolysis of 6-phospho-␣-glucoside substrates including maltose 6-phosphate and trehalose 6-phosphate. In metal-free buffer, GlvA exists as an inactive dimer, but in the presence of Mn 2؉ ion, these species associate to form the NAD(H)-dependent catalytically active tetramer. By comparative sequence alignment with its homologs, the novel 6-phospho-␣-glucosidase from B. subtilis can be assigned to the nine-member family 4 of the glycosylhydrolase superfamily.The serendipitous discovery in 1964 (1, 2) of the bacterial phosphoenol pyruvate-dependent sugar phosphotransferase system (PEP-PTS) 1 by Roseman and colleagues represents a landmark in our understanding of carbohydrate transport by microorganisms (3, 4). Since the initial description in Escherichia coli, this phosphoryl group-transfer system (5, 6) has been established as the primary mechanism for the accumulation of sugars by bacteria from both Gram-negative (7, 8) and Gram-positive genera (9 -12). Operationally, the multi-component PEP-PTS (13) comprises both membrane-localized and cytoplasmic proteins that in concert catalyze the simultaneous phosphorylation and vectorial translocation of sugar across the cytoplasmic membrane. Catalytically, each PEP-PTS requires two general components (Enzyme I and HPr) that, allied with sugar-specific proteins (IIA, -B, and -C; for discussion, see Ref. 14), promote the sequential transfer of the high energy, phosphoryl moiety from PEP to the incoming sugar. Prior to catabolism via energy-yielding pathways, the intracellular disaccharide phosphates must first be hydrolyzed to their constituent hexose 6-phosphate and aglycone moieties. Several phosphoglycosylhydrolases (whose ge...

Metabolism of sucrose and its five isomers by Fusobacterium mortiferum

“…As reported for other members of this unusual family, MalH is inherently unstable and Mn# + and NAD + are prerequisite cofactors for activity (Nagao et al, 1988 ;Thompson et al, 1998Thompson et al, , 1999Raasch et al, 2000). Whether the nucleotide and metal ion fulfil catalytic or structural functions has not been ascertained for any member of family 4.…”

Genes malh and pagl of Clostridium acetobutylicum ATCC 824 Encode NAD+- and Mn2+-dependent Phospho-α-glucosidase(s)

“…Identical amino acids are highlighted (black background), and numbers at left and right denote residue positions. The arrows above the sequences at the N terminus indicate a portion of the nucleotide-binding domain, including the conserved G(X)GS motif of GHF4 proteins (14,16,18,19). Arrows beneath the sequences indicate residues that constitute the signature pattern of proteins in this family.…”

“…Arrows beneath the sequences indicate residues that constitute the signature pattern of proteins in this family. 1 Glutamyl residues that may participate in catalysis (14,19) are indicated by asterisks. The filled circle shows the cysteine residue that is positionally conserved in all GHF4 proteins, and that occurs in the CDMP motif of the phospho-␣-glucosidase subgroup of enzymes.…”

“…The GHF4 enzymes have received considerable attention, in part, because members of this unique family are distinguished from all others by their requirements for reducing agent, divalent metal ion (Mn 2ϩ , Co 2ϩ , Ni 2ϩ , or Fe 2ϩ ) and dinucleotide (NAD ϩ ) for activity. Whether these cofactors function in a catalytic or structural capacity in GHF4 enzymes has yet to be established (14,18,19).…”

“…30 two genes for which the deduced open reading frames exhibited extensive homology with an NAD ϩ and metal-dependent GHF4 phospho-␣-glucosidase that we had found in several bacterial species, including Bacillus subtilis (14), Fusobacterium mortiferum (13,23,24), and Klebsiella pneumoniae (15). For purposes of distinction, we refer to the two putative phospho-␣-glucosidases in C. acetobutylicum 824 as MalH (maltose 6-phosphate hydrolase) and PagL (phospho-␣-glucosidase), respectively.…”