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V M Lam

Publications and source records attributed to V M Lam.

18 recordsLinked to original sources
PubMed2000

Human glucose-6-phosphate dehydrogenase: the crystal structure reveals a structural NADP(+) molecule and provides insights into enzyme deficiency.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) catalyses the first committed step in the pentose phosphate pathway; the generation of NADPH by this enzyme is essential for protection against oxidative stress. The human enzyme is in a dimer<-->tetramer equilibrium and its stability is dependent on NADP(+) concentration. G6PD deficiency results from many different point mutations in the X-linked gene encoding G6PD and is the most common human enzymopathy. Severe deficiency causes chronic non-spherocytic haemolytic anaemia; the usual symptoms are neonatal jaundice, favism and haemolytic anaemia. RESULTS: We have determined the first crystal structure of a human G6PD (the mutant Canton, Arg459-->Leu) at 3 A resolution. The tetramer is a dimer of dimers. Despite very similar dimer topology, there are two major differences from G6PD of Leuconostoc mesenteroides: a structural NADP(+) molecule, close to the dimer interface but integral to the subunit, is visible in all subunits of the human enzyme; and an intrasubunit disulphide bond tethers the otherwise disordered N-terminal segment. The few dimer-dimer contacts making the tetramer are charge-charge interactions. CONCLUSIONS: The importance of NADP(+) for stability is explained by the structural NADP(+) site, which is not conserved in prokaryotes. The structure shows that point mutations causing severe deficiency predominate close to the structural NADP(+) and the dimer interface, primarily affecting the stability of the molecule. They also indicate that a stable dimer is essential to retain activity in vivo. As there is an absolute requirement for some G6PD activity, residues essential for coenzyme or substrate binding are rarely modified.

Amino Acid Sequence↗
PubMed1999

Solution of the structure of tetrameric human glucose 6-phosphate dehydrogenase by molecular replacement.

Recombinant human glucose 6-phosphate dehydrogenase (G6PD) has been crystallized and its structure solved by molecular replacement. Crystals of the natural mutant R459L grow under similar conditions in space groups P212121 and C2221 with eight or four 515-residue molecules in the asymmetric unit, respectively. A non-crystallographic 222 tetramer was found in the C2221 crystal form using a 4 A resolution data set and a dimer of the large beta + alpha domains of the Leuconostoc mesenteroides enzyme as a search model. This tetramer was the only successful search model for the P212121 crystal form using data to 3 A. Crystals of the deletion mutant DeltaG6PD grow in space group F222 with a monomer in the asymmetric unit; 2.5 A resolution data have been collected. Comparison of the packing of tetramers in the three space groups suggests that the N-terminal tail of the enzyme prevents crystallization with exact 222 molecular symmetry.

Amino Acid Sequence↗
PubMed1996

Rapid detection of common Chinese glucose-6-phosphate dehydrogenase (G6PD) mutations by denaturing gradient gel electrophoresis (DGGE).

We describe here the use of denaturing gradient gel electrophoresis (DGGE) to detect the most common Chinese glucose-6-phosphate dehydrogenase (G6PD) variants, which are the single point mutations: G-->T at nt 1376, G-->A at 1388 both in exon 12 and A-->G at nt 95 in exon 02. In each case, the mutant allele resolves well from the normal allele(s). The distinct heteroduplex bands are characteristic of a particular genotype suggesting that this feature is very useful for identifying all heterozygous carriers for this and other X-linked diseases. When the analysis is extended to other exons, DGGE scans the gene and coupled with direct sequencing, it leads to the identification of new G6PD variation(s). With this approach, we identified a mutation in exon 9 which had not been reported in Hong Kong. Since DGGE can rapidly screen many unknown samples in one gel, this approach could be used to diagnose these G6PD mutations and to identify the at-risk for counselling.

Alleles↗
PubMed1995

Genomic organization and characterization of a three-gene rat adult beta-globin haplotype.

The isolation and detailed characterization of a three-beta-globin gene (GloB) haplotype in the Sprague-Dawley (S-D) rat is described. An enriched library, lambda SDHelib, was screened with a human GloB probe, humbg44, and from which a beta minor gene, Rathbbz, was isolated, sequenced and characterized. A S-D rat GloB-specific probe, Ratbgze12, derived from the Rathbbz gene, was then used to screen a S-D rat genomic library, lambda SDglib. The clone T1510 was isolated and identified to include the entire Rathbbz gene and part of another GloB gene, Rathbby, which was 5' upstream from Rathbbz. Chromosomal walking upstream using the riboprobe, rnaT71, led to the isolation of an overlapping clone, Ta49, which was shown to include two full-length GloB genes; the most 5' was Rathbbx followed by Rathbby. Sequence data suggests that Rathbbx is a beta major gene, whereas Rathbby is a hybrid gene of Rathbbx and Rathbbz. Genomic hybridization confirmed this particular three-gene haplotype in the S-D rat. This haplotype, a1, may be the prototype of the GloB cluster in rat.

Animals↗
PubMed1993

Two new rat alpha-globin sequences as identified by the conserved region PCR.

Based on the notion that regions of structural genes which encode critical domains of the corresponding proteins are highly conserved among closely related species, oligonucleotide primers were designed and used to amplify the alpha-globin sequence(s) of the Sprague-Dawley (SD) rat. Data of these amplified sequence constructs showed that two new rat alpha-globin specific sequences have been identified. Southern hybridization confirmed the presence of these sequences in the rat genome.

Animals↗
PubMed1991

Mapping irradiation hybrids to cosmid and yeast artificial chromosome libraries by direct hybridization of Alu-PCR products.

A direct hybridization protocol is described for screening cosmid and yeast artificial chromosome libraries with pools of Alu-PCR products from somatic cell or irradiation hybrids. This method eliminates purification, cloning and analysis of each individual Alu-PCR product before library screening. A series of human X chromosome irradiation hybrids were mapped by this method, using a cosmid reference library for comparisons between overlapping hybrids to identify interesting clones for further analysis.

Base Sequence↗
PubMed1988

Molecular characterization of beta-globin gene mutations in patients with beta-thalassaemia intermedia in south China.

We have studied the spectrum of mutations producting beta-thalassaemia intermedia in South China. The methods of mutation detection include oligonucleotide analysis, polymerase chain reaction amplification of the beta-globin gene and direct genomic sequencing. The mutations have been identified in 22 beta-globin genes from the patients in 11 unrelated families. Seven different mutations have been identified and the A to G substitution in the TATA box of the beta-globin gene accounts for 42% of these mutant beta-globin genes. Most patients have a beta(+) thalassaemia and one copy of the TATA box mutation. In two patients with beta(0) thalassaemia intermedia the mild phenotype may be explained in one by the presence of the - + - + + 5' beta-globin gene cluster haplotype which contains the Xmn I site -158 nt to the G gamma-globin gene or in the other by the number of alpha-globin genes present.

Base Sequence↗
PubMed1980

Role of L-threonine deaminase and L-threonine 3-dehydrogenase in the utilization of L-threonine by Pseudomonas aeruginosa.

Mutants of Pseudomonas aeruginosa PAC1 which could grow on L-threonine were isolated. These mutants, like the parent strain, synthesized a biosynthetic threonine deaminase, but its apparent Km value for threonine was higher than that of the enzyme from strain PAC1. These mutants also synthesized an inducible NAD-dependent threonine dehydrogenase, which was not present in the parent strain. No threonine aldolase activity could be detected. The results suggest that the threonine deaminase with lowered affinity for L-threonine, together with L-threonine dehydrogenase, enabled these mutants to utilize L-threonine as the sole source of carbon for growth.

Alcohol Oxidoreductases↗
PubMed1980

Proton-linked D-xylose transport in Escherichia coli.

The addition of xylose to energy-depleted cells of Escherichia coli elicited an alkaline pH change which failed to appear in the presence of uncoupling agents. Accumulation of [14C]xylose by energy-replete cells was also inhibited by uncoupling agents, but not by fluoride or arsenate. Subcellular vesicles of E. coli accumulated [14C]xylose provided that ascorbate plus phenazine methosulfate were present for respiration, and this accumulation was inhibited by uncoupling agents or valinomycin. Therefore, the transport of xylose into E. coli appears to be energized by a proton-motive force, rather than by a phosphotransferase or directly energized mechanism. Its specificity for xylose as inducer and substrate and the genetic location of a xylose-H+ transport-negative mutation near mtl showed that the xylose-H+ system is distinct from other proton-linked sugar transport systems of E. coli.

Arsenates↗