Die Lipase Engineering Database : systematische Analyse familienspezifischer Eigenschaften und der Sequenz-Struktur-Funktionsbeziehung von alpha/beta-Hydrolasen

Abstract

Im Rahmen dieser Arbeit wurde die Familie der alpha/beta-Hydrolasen systematisch untersucht. Grundlage dieser Analyse war die Entwicklung eines Data Warehouse Systems zur Integration von Proteinsequenz- und Strukturdaten in Verbindung mit funktionell relevanten Informationen für große Proteinfamilien. Als Anwendung dieses Data Warehouse Systems wurde die Lipase Engineering Database (LED) erstellt, die alle Mitglieder der alpha/beta-Hydrolasen enthält. Das Release 2.3 der LED enthält 3148 Sequenzeinträge für 2313 Proteine, wobei 35% der Proteine als putativ definiert sind. Für 96 Proteineinträge sind 261 Struktureinträge in der LED abgelegt. Aufgrund der Sequenzähnlichkeit wurden die alpha/beta-Hydrolasen in 37 Superfamilien und 103 homologe Familien eingeteilt. Die LED diente zur systematischen Identifikation familienspezifischer Deskriptoren auf Sequenz- und Strukturebene. Die Bestimmung streng konservierter Positionen für acht repräsentative alpha/beta-Hydrolasesuperfamilien half bei der Identifikation des Faltungsnukleus, der den alpha/beta-Hydrolase Fold charakterisiert. Daraus konnte ein Modell für die Faltung dieser Proteine skizziert werden. Anhand der Architektur des funktionell wichtigen oxyanion holes konnten diese in drei Klassen eingeteilt werden: (1) die GGGX Klasse, für die die oxyanion hole bildende Aminosäure am C-terminalen Ende des hoch konservierten Musters GGG lokalisiert ist und von einer streng konservierten, hydrophoben Aminosäure X gefolgt wird, (2) die GX Klasse, für die die oxyanion hole Aminosäure X einem streng konserviertem Glycin folgt und (3) die Y Klasse deren oxyanion hole durch die Hydroxylgruppe eines streng konservierten Tyrosin gebildet wird. Für die strukturelle Stabilisierung des oxyanion holes der GX Klasse wurde die Wechselwirkung der Seitenkette X mit Anker Aminosäuren identifiziert. Dieses Anker-Konzept konnte experimentell durch den Austausch des Anker-Moduls in der Lipase aus Burkholderia cepacia gegen das der Lipase aus Rhizomucor miehei bestätigt werden. Die Form und die physikalisch-chemischen Eigenschaften der Fettsäurebindungsstelle von acht alpha/beta-Hydrolasen wurden untersucht, um deren Substratspezifität auf molekularer Ebene nachzuvollziehen. Diese konnten so in vier Gruppen eingeteilt werden und half bei der Identifikation von Aminosäuren, die für die Vermittlung der Substratkettenlängenspezifität verantwortlich sind. Für die Superfamilie der Carboxylesterasen wurden mit der in CODEHOP implementierten Methode degenerierte familienspezifischen Primer erstellt, die sich zur Identifikation neuer Carboxylesterasen in aus Bodenproben isolierter DNA eignen.

To facilitate the systematic analysis of large protein families a data warehouse system to store and analyze sequence, structure, and functional annotation information was developed. This data warehouse system was applied to set up the Lipase Engineering Database (LED) which includes all members of the alpha/beta hydrolase fold family. The alpha/beta hydrolases represent one of the largest families in protein space of structurally related proteins. In the release 2.3 the LED includes 3148 sequences of 2313 proteins entries, 35% of them being putative proteins. For 96 protein entries 261 structure data sets from the Protein Databank were stored. Proteins were assigned to 37 superfamilies and 103 homologous families. The sequence annotation validation resulted in the assignment of the catalytic triad for all superfamilies, for which structure information was available. By sequence comparison, these functional residues were assigned in 25% of homologous families and 53% of all sequence entries. The LED has been applied to systematically analyze the alpha/beta hydrolase fold family. Based on sequence and structure data family specific descriptors were inferred, the effect of mutations could be explained, new functionally relevant modules were identified and substrate specificity could be predicted by the gene sequence. By analyzing the amino acid conservation for eight superfamilies the mutually conserved residues were identified. These residues are conserved within families, but show differences between families though they are located at equal positions within a common protein fold. These positions are claimed to form the folding nucleus. For the alpha/beta hydrolase fold 54 mutually conserved residues were identified. Based on this findings a model for the process of folding of the alpha/beta hydrolases was postulated. To estimate the relationships between the superfamilies of alpha/beta hydrolases, Hidden Markov Models (HMM) were generated for each homologous family. Distances were inferred by profile HMM-HMM comparison and used to create a phylogenetic tree. According to this tree a systematic nomenclature for alpha/beta hydrolases was introduced. Based on structure and sequence analysis of the oxyanion hole, alpha/beta hydrolases were classified into three classes, the GGGX-, GX- and Y-class. The GX class consists of 20 superfamilies with known protein structures, where the oxyanion hole forming residue X is structurally well conserved and was preceded by a strictly conserved glycine. The GGGX class consists of five superfamilies with known protein structures, where the oxyanion hole forming residue is located in a well conserved GGG pattern, which is followed by a conserved hydrophobic amino acid X. The backbone amide of glycine G preceding the residue X is forming the oxyanion hole. The Y class consists of four superfamilies with known protein structures, where the oxyanion hole is not formed by a backbone amide, but by the hydroxyl group of a tyrosine side chain, which is strictly conserved within the superfamilies. To understand the stabilization of the structurally well conserved oxyanion hole architectures, sequence and structure data were compared and analyzed. For the GX class the side chain of X is stabilized by one or several anchor residues, which are also well conserved within the families. The conservation of the oxyanion hole residue and the anchor within the families indicated a modular organization of the first oxyanion hole residue and the anchor. To proof the modularity of the first oxyanion hole residue and the anchor of the GX class the hydrophobic module of the Lipase from Burkholderia cepacia (BCL) was exchanged for the hydrophilic module of the Lipase from Rhizomucor miehei (RML) in two steps. While the replacement of the oxyanion hole residue resulted in a 200 fold decrease in activity the subsequent exchange of the anchor L167 with an asparagine resulted in the reactivation of the BCL, proofing the anchor concept. Shape and physico-chemical properties of the scissile fatty acid binding sites of eight alpha/beta hydrolases were analyzed and compared in order to understand the molecular basis of substrate specificity. The alpha/beta hydrolases were divided into four subgroups by characterizing the location and the properties of the scissile fatty acid binding sites. Thus the residues which mediate chain length specificity could be identified. To utilize the LED for identifying new members of the alpha/beta hydrolase fold family from genomic DNA or soil probes family specific PCR primers were designed. A set of degenerated primers were inferred for a superfamily including short chain length specific lipases and carboxylesterases. After validating the specificity of these primers using Bacillus subtilis as model organism, miscellaneous organisms were screened successfully for carboxylesterases.